Atomic structure of the hemalbumin complex and its use in designing therapeutic

ABSTRACT

A high resolution structure of the hemalbumin binding complex is provided which includes the detailed atomic coordinates which reflect the binding site and the binding characteristics of the structure. This high resolution structure can be used in methods of determining the primary residues involved in gas binding, redox potential of iron, etc., and thus will be used to identify and optimize the gas binding characteristics of heme and albumin, so as to allow for the development of modified recombinant albumins containing heme and/or heme derivatives which have improved gas binding properties and which can be used for therapeutic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/361,074, filed Mar. 1, 2002.

FIELD OF THE INVENTION

This invention relates in general to a high resolution crystal structureof hemalbumin, and in particular to the atomic coordinates for thebinding region between serum albumin and heme, methods of obtainingthose coordinates, and methods of using said coordinates to obtain vitalinformation regarding potential binding sites in albumin and heme so asto enable the development of genetically modified versions of thesemolecules with improved binding properties and therapeutic usefulness.

BACKGROUND OF THE INVENTION

The serum albumins are the major soluble proteins of the circulatorysystem and contribute to many vital physiological processes. Serumalbumin generally comprises about 50% of the total blood component bydry weight, and as such is responsible for roughly 80% of themaintenance of colloid osmotic blood pressure and is chiefly responsiblefor controlling the physiological pH of blood. The albumins also play anextremely important role in the transport, distribution and metabolismof many endogenous and exogenous ligands in the human body, including avariety of chemically diverse molecules including fatty acids, aminoacids, steroids, calcium, metals such as copper and zinc, and variouspharmaceutical agents. The albumins are generally thought to facilitatetransfer many of these ligands across organ-circulatory interfaces suchas the liver, intestines, kidneys and the brain, and studies havesuggested the existence of an albumin cell surface receptor. See, e.g.,Schnitzer et al., P.N.A.S. 85:6773 (1988). The albumins are thusintimately involved in a wide range of circulatory and metabolicfunctions.

Human serum albumin (HSA) is a protein of about 66,500 kD protein and iscomprised of 585 amino acids including at least 17 disulfide bridges. Aswith many of the albumins, human serum albumin plays an extremelyimportant role in human physiology and is located in virtually everyhuman tissue and bodily secretion. As indicated above, HSA has anoutstanding ability to bind and transport and immense spectrum ofligands throughout the circulatory system including the long-chain fattyacids which are otherwise insoluble in circulating plasma. Certaindetails regarding the atomic structure and the binding affinities ofalbumin and the specific regions primarily responsible for those bindingproperties have previously been disclosed, e.g., in U.S. patentapplication Ser. No. 08/448,196, filed May 25, 1993, now U.S. Pat. No.5,780,594 and U.S. patent application Ser. No. 08/984,176, filed Dec. 3,1997, now U.S. Pat. No. 5,948,609, both of which are incorporated hereinby reference.

Because of the vital role played by albumins, there are literallythousands of applications for serum albumin covering a wide range ofphysiological conditions. However, unlike blood proteins such ashemoglobin, native serum albumins are non-functional as oxygen transportsystems, and thus have not been useful in blood replacement systemsrequiring oxygen transport. Accordingly, one recent focus of researchhas been the binding of the albumin molecule with heme, one of theimportant blood proteins. Under normal physiological conditions, hemethat finds its way into plasma is bound by the specific heme-bindingprotein, hemopexin, which delivers it to the liver for excretion via areceptor-mediated uptake mechanism (1-5). Under pathophysiologicalconditions of severe hemolysis when significant amounts of freehemoglobin appear in the circulation, serum albumin can also become asignificant transporter of heme (6,7), principally as hemin (Fe^(III)Protoporphyrin-IX (Cl)). These are conditions when hemopexin becomessaturated by hemin, and albumin, which is present at considerably higherconcentration than hemopexin, acts as a depot for the overflow.Additionally, a source of heme uptake by albumin has been suggested toresult from the uptake of soluble heme-containing peptides released bythe enzymatic digestion of dietary heme-containing proteins such ascytochrome c, where they may constitute a significant route by whichiron enters the mammalian system (8).

Hemin is one of the important endogenous ligands transported and/orsequestered by human albumin and among the most highly bound having witha predicted a single high affinity site with K_(A)=1.1×10⁸ M⁻¹ (9).Interestingly, among mammals, only albumin of primates shows a singlehigh affinity heme binding site (4). Studies of heme binding to albuminsuggest a two step binding process, a fast interaction to form anintermediate complex, followed by “internalization” of the hemin in aregion with limited access to bulk aqueous solvent (9,10). Althoughvarious hypotheses concerning the binding location and chemistry tohuman albumin have been proposed from spectroscopic and other methods(11-17), except for the general binding location within cleavagefragments (IB-IIA) (6) and more recently recombinant domains (domain I)(18), the conclusions of all of the other studies are inconsistent withthe location and coordination of the atomic structure of the complex inaccordance with this invention as described herein.

Previously, the structure of human methemalbumin was determined at 2.8 Å(19) using a crystal form of the space group C2, i.e. form-III asreported in reference (20). These publications evidenced that the hemebinds to albumin within the IB pocket, a site that was previouslyidentified with long chain fatty acid transport. In addition, asuccessful blood replacement product was obtained which featured aheme-albumin complex with a recombinant serum albumin having at leastone of the four key hydrophobic binding residues in the heme bindingregion replaced with a histidine, such as disclosed in U.S. Pat. No.5,948,609, incorporated herein by reference. However, the ability touncover additional information which would lead to further breakthroughswith regard to the capacity of albumin to be utilized to further improvegas binding properties has heretofore been limited by the resolution ofthe crystal albumin structure obtainable. Similarly, it is alsoimportant to obtain additional insights with regard to the heme moleculeand its derivatives so as to be able to develop heme molecules andderivatives which when combined with albumin will provide oxygen orother gas binding/transport delivery properties or allow for otherapplications such as scavenging of toxic gases (e.g., cyanide, nitricacid, carbon monoxide, etc.).

Accordingly, it is highly desirable to develop a system of determiningkey binding regions of the heme/albumin complex and to develop a meansby which the alteration of albumin or heme genetically can beaccomplished so as to identify and maximize the medically relevant gasbinding properties of these molecules, a goal that has not previouslybeen achievable with lower resolution pictures of the albumin crystalcomplex structure at the relevant gas binding sites.

SUMMARY OF THE INVENTION

Accordingly, it is thus an object of the present invention to provide ahigher resolution picture of albumin at the hemalbumin binding complexso as to be useful in determining novel sites which will be used toidentify and maximize albumin gas binding characteristics.

It is further an object of the present invention to provide a method fordeveloping high resolution atomic coordinates of the heme/albumincomplex that will allow the design of therapeutics based on the complexand allow for the development of modified recombinant albumins whichhave improved gas binding properties.

It is further an object of the present invention to provide a method forguiding the development of novel small molecule heme derivatives whichwhen combined with albumin will have maximized gas-binding properties soas to be useful in a wide variety of applications including delivery ofoxygen, binding, transport or delivery of other gases, and scavenging ofpotentially toxic gases (e.g., cyanide, nitric acid, carbon monoxide,etc.).

It is still further an object of the present invention to provide highresolution atomic coordinates of the heme/albumin complex that willallow the design of therapeutics based on the complex and allow for thedevelopment of modified recombinant albumins which have improved gasbinding properties.

These and other objects of the present application are obtained byvirtue of the present invention which comprises a method of obtaininghigh resolution atomic coordinates of the heme/albumin complex, andwhich provides the atomic coordinates thereof in a manner not previouslypossible. The provision of the high resolution atomic coordinates ofthis crystal complex in accordance with the invention allows for theidentification of key binding sites and will thus allow for thedevelopment of genetically modified albumin and heme molecules whichhave maximized gas binding properties and which can be useful in manyapplications such as binding, transport, and delivery or therapeuticgases such as oxygen, and scavenging and removal of potentially toxicgases such as cyanide, nitric acid, carbon monoxide, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated in the drawings, wherein:

FIG. 1 is a stereo view illustrating the difference density for thehemin (F_(obs)-F_(calc)) shown contoured at 3σ after a simulatedannealing refinement without the incorporation of heme into the model.The heme coordinates from the final refined structure are shownsuperimposed on the initial difference density. The protrusion of the Feelectron density toward the proximal side indicating a position abovethe heme plane in the direction of Tyr-161, can be clearly seen.

FIG. 2 is a stereo view of the C_(α) carbon tracing of subdomain IB(red) illustrating the helical motif (helices h7 through h10) and thebinding location of the heme (green) in the binding pocket.Additionally, the striking overlap of the binding site with myristate(yellow) is shown for comparison.

FIG. 3A is a stereo view from the ‘proximal’ side of the heme showinganother perspective of the coordination of the Tyr-161 hydroxyl with theFe atom and the salt bridge and hydrogen bonding interactions ofLys-190, His-146, and Arg-114. The water molecules located in thebinding pocket and associated with Tyr-161 which have been discussed inthe text are illustrated.

FIG. 3B is a stereo view of the heme pocket as viewed from the surfaceopening. Close interactions are seen with Ile 142 (right or ‘distal’side), Tyr-161 (left or ‘proximal’ side) and Tyr 138 (top right) withinthe pocket and the salt bridges with Lys-190, the ‘gate’ residue andHis-146. Note the water coordination and close proximity of Arg-114 tothe heme carboxylate (top right).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, there are provided atomiccoordinates for the heme/albumin binding complex (or “hemalbumin”) whichis obtained as a high resolution structure as determined by singlecrystal X-ray diffraction to a resolution of 1.9 Angstroms. As describedfurther below, The structure as determined in the present inventionrevealed that protoporphyrin IX was bound to a single site within ahydrophobic cavity in subdomain IB, one of the principal binding sitesfor long chain fatty acid. This iron is penta coordinated with the fifthligand comprised of the hydroxyloxygen of Tyr-161 (phenolic oxygen toheme plane distance: 2.73 Å) in an otherwise completely hydrophobicpocket. The heme propionic acid residues form salt bridges with His-142and Lys-190, which together with a series of hydrophobic interactions,enclose and secure the hem within the IB helical motif. A detaileddescription of the structure together with its implications for thedevelopment of potential blood substitutes is provided hereinbelow.

As explained further in U.S. Pat. No. 5,948,609, the initial complex ofheme and albumin subject to the high resolution of the present inventionmay be obtained in a number of suitable ways, such as by dissolvinghemin and combining it with serum albumin such as a natural orrecombinant serum albumin such as described above. As will be set forthbelow, the albumin used may be one in its unmodified state so as todetermine the precise nature of the key residues involved inheme/albumin binding. Further, in accordance with the invention, heminmay be combined with a serum albumin that has been modified at certainkey residues so as to enhance gas binding properties, and the highresolution of such structures in accordance with the invention canassist in determining if such modifications result in improved bindingcomplexes. Heme, or protoporphyrin, is one of the many metabolicproducts of endogenous origin and is produced from myoglobin andhemoglobin. In general, it appears that only the albumins from primateshave a specialized high affinity site for protoporphyrin, and bindingconstants for this ligands are on the order of 1×10⁸ Ka. In accordancewith the invention, crystals of human serum albumin have been complexedwith heme and studied using high resolution crystallographic means whichhave provided the atomic coordinates needed to investigate atomicinteractions between albumin and heme and use the information to bettermodel these molecules for maximization of binding properties.

In order to utilize crystallographic processes to study the bindingbetween heme and albumin, hemin was dissolved and combined with humanserum albumin in a 1:1 Molar ratio, which produces methemalbumin whichis also crystallized utilizing conditions such as those disclosed inCarter et al., 1994 and co-pending U.S. patent application Ser. No.08/448,196, incorporated herein by reference. X-ray diffraction datawere collected and subsequent printouts produced from these dataprovides precise information concerning the binding location andchemistry of the heme/albumin complex.

In the particularly preferred embodiment, the hemalbumin crystals may beprepared in a manner previously described (19,20). The human albumin isdefatted in the manner described in Chen (21) and is preferably coupledwith 1:1 molar ratio of albumin:hemin (Fe^(III)-Protoporphyrin-IX (Cl))prior to coupling with myristate, although this is not always required.The crystals are of the same C2 form as the form-III of reference (19).Diffraction data were collected at the Beamline 5.0.3 of the AdvancedLight Source (ALS), Lawrence Berkeley Laboratory (LBL). A 2×2 array(ADSC) of CCD detectors was used with a detector-to-crystal distance of180 mm. A total of 180 (1°) oscillation images were collected from onecrystal at 100 K using photon wavelength of 1.0 Å and exposure of 60second per frame. At the cryogenic temperature, the unit cell dimensionswere a 183.11 Å, b=37.91 Å, c=94.83 Å, β=105.04°. These images yielded adata set of 44,335 independent reflections with positive intensities toa resolution of 1.9 Å (R_(merge): 3.7%; average multiplicity: 2.03;average l/σ(1): 14.7, and completeness: 88.1%).

The program package of CNX X-ray from Accelrys, Inc. was used forstructure refinement, with the protein starting model taken from astructure of human albumin complexed with myristate (without hemin)determined in our earlier studies (19,22) and re-refined more recentlyat 100 K. The hemin and five myristate molecules were clearly resolvedin the difference density maps after initial refinement. When hemin,myristates, and solvent water molecules were incorporated in the model,the refinement quickly converged to an R-factor of 22.8% for all 42,107reflections in the working set with no sigma cut-off (R-free 28.2%),yielding a structure of good geometry giving rms deviations fromideality of 0.005 Å for bond-distance and 1.19° for valence-angle. Thefinal model includes 583 protein residues, 1 hemin molecule, 5 myristicacid molecules and 581 solvent water molecules with an average B-factorof 27.8 Å² for the protein atoms. One residue each at both N- andC-termini were not visible on the electron density maps, presumablydisordered, and were not included in the model. Atomic coordinates ofthe methemalbumin structure at the binding region IB are included hereinas Appendix A.

In accordance with the present invention, a hemalbumin structure waswell determined and represents the highest resolution albumin structurereported to date, and details of the refinement statistics are providedin Table I included below. The quality of the resulting electron densityis high providing detailed information of the heme binding interactionas well as other important structural information such as positions ofkey water molecules. The orientation of the heme(Fe^(III)-Protoporphyrin-IX) provides a good fit to the electron densityas shown in FIG. 1. There is the possibility that a mixture of the twoorientations actually exists in the complex (180 degree rotationalisomers along the CHA to CHC line). However, the orientation chosenprovides the best fit, and neither orientation affects the resultinginterpretation of the binding chemistry. The overall structure ofmethemalbumin is very similar to the HSA-myristate structure aspreviously determined and used in this work to derive the phases for themethemalbumin structure (discussed above); and the structure using thesame crystal forms and reported by others (23). The structures have anrms deviation between Cα atom of positions of 0.396 Å. When Cα forresidues 109-195 that constitute the IB subdomain are superimposed, therms was 0.32 Å.

A single site for hemin revealed by earlier lower resolution studies(19) was verified in accordance with the high resolution form of thepresent invention. The hemin is located within the IB subdomain which isconstituted by a loop and four contiguous helices (h7, h8, h9 and h10)(22,24). The heme is buried in a hydrophobic cleft or pocket formed bythe subdomain helices and its position within IB forms a strikingsuperposition on the plane represented by the curved structure assumedby long chain fatty acid when occupying this site (FIG. 2). One half ofthe pocket is enclosed by h9 and h10, the other half by the loop h8, thetop by h7 and the bottom by the loop between h8 and h9. The plane of theinserted heme lies approximately 30 degrees to the helical axes of h9and h10.

In accordance with the invention, the high resolution crystal structureallowed for the elucidation of the heme binding region in albumin andrevealed the key binding regions in this area. In particular, there arefive residues that show close interaction with the heme and appear to bekey contributors to the high binding affinity, Tyr-161, Ile-142,Tyr-138, His-145, and Lys-190. The tyrosine side chains form closeparallel stacking interactions with the heme (FIG. 3). One of the keyheme interactions involves Tyr-161, which shows coordination of thephenolic oxygen with the heme iron resulting in an oxygen to heme planedistance of 2.73 Å and a pentacoordinate iron. This phenolic oxygenreplaces the chloride ion of the hemin complex in solution(Fe^(III)-Protoporphyrin-IX (Cl)). The details of the methemalbumin hemeare similar to the original hemin small molecule structure determined in1965 (25) where: 1) the iron is located off the heme surface (0.475 Å)(the Fe electron bulge in the methemalbumin indicates a similarmovement, see FIG. 1); 2) the Chloride to heme plane distance is 2.693 Å(methemalbumin Tyr-161 O to heme plane distance 2.73 Å); and 3) hemepotentially disordered (averaged) by the two hemes rotated 180° about aline between the CHA and CHC atoms in both structures. In themethemalbumin structure the Fe atom was restricted to co-planarity withthe heme during refinement, however, the difference maps as in FIG. 1,show a significant deviation from planarity, suggesting an iron positionsimilar to the original hemin small molecule structure. If one assumesthe iron atom also lies 0.475 Å off the heme surface in methemalbumin,then the Tyr-161 phenyl oxygen to iron distance can be estimated at 2.27Å, a value in good agreement with the Cl to Fe distance of 2.22 Å inhemin (25). In the more recent malarial pigment β-hematin structurewhere the carboxylate oxygen of a symmetrically related heme becomes thefifth coordinate to the Fe atom, the O to Fe distance was determined at1.89 Å (26).

In addition to the coordination with the heme iron, the phenolic oxygenof Tyr-161 forms hydrogen bonds with a series of water molecules (W1058,W1099, and W1244) within the hydrophobic cavity and extending into awell defined surface water structure (FIG. 3B). In accordance with theinvention, this side of the heme is referred to as the “proximal” side.No water molecules were observed on the opposite or ‘distal’ side of theheme pocket. The propionic residues of the heme protrude from the pocketand form two important salt bridges, one with His-145 which forms astrong bridge with the ‘A’ ring carboxylate and the other with Lys-190,which forms a salt bridge mid way between both the ‘A’ and ‘D’ ring hemecarboxyls (FIG. 3). Ile-142, near the iron on the distal side of theheme, contributes one of the closest hydrophobic interactions.

The residues having close interaction or contributing to the hydrophobicsurface of the binding pocket include: Tyr-161, Phe-157, Arg-186,Leu-182, Arg-117, Phe-134, Leu-135, Leu-154, Phe-149, Ile-142, His-146,Arg-114, Lys-190, Ser-193, Ala-158, Tyr-138, Leu-115, Met-123, Phe-165,and Pro-118. Details of many of the heme binding interactions of theseresidues are illustrated in FIG. 3A. In accordance with the invention, arecombinant serum albumin with improved properties can be preparedwherein certain residues contributing to the hydrophobic surface of thebinding pocket are replaced with residues having a greater affinity toheme. For example, improved binding properties may be obtained whereinat least one of the these key hydrophobic binding residues in the hemebinding region replaced with a hydrophilic binding residue, such ashistidine. In addition, the creation of optimal performance may alsorequire the simultaneous replacement of two or more of the definedresidues with residues of different charge or hydrophobicity, therebyimproving the gas affinity and oxidation rates. These recombinantalbumins can be prepared in accordance with the invention in anysuitable manner well known in the art including those techniques as setforth in the prior patents and applications set forth above, e.g.,wherein nucleic acid coding for the desired mutation is expressed usingan appropriate expression vector.

Comparisons of the hemalbumin structure with the nativealbumin/myristate structure reveal four residues that show pronouncedmovements upon heme complexation. The first of these is Ile-142 wherethe CD1 atom has swung about 135° away from the heme plane to avoidsteric clash, resulting in a CD1 atom within 3.38 Å from the NAnitrogen. His-146 displays perhaps the largest movement uponcomplexation, making a key salt bridge with the ‘A’ ring carboxylate ofheme, resulting in an equidistant His NE atom to O1A/O2A distance of3.30 Å. The third residue demonstrating significant side chain movementis Lys-190. In the HSA-myristate structure, the NZ nitrogen at the endof the side chain of Lys-190 makes a 2.99 Å salt bridge with the OD1oxygen of Asp-187. In the methemalbumin structure, this salt bridge isbroken resulting in the 180° rotation of the Lys-190 side chain aboutits CD atom across the pocket opening to form a key salt bridgeinteraction with both the heme carboxyls. This new position wassupported by clear difference electron density. In this position, the NZnitrogen atom of Lys-190 is almost midway between the two hemecarboxylate groups and makes a salt bridge with each of them. Theshortest (2.99 Å) of these is made with the O2D oxygen of the heme ‘D’ring carboxylate, while a slightly longer salt bridge (3.32 Å) is formedbetween the NZ atom of Lys-190 and the O1A oxygen of the heme ‘A’ ringcarboxylate. The swinging motion by the side chain of Lys-190 can belikened to a gate shutting after the hemin/heme is bound with the saltbridges serving as a latch to keep the gate shut. Arg-114, the fourthresidue, has its side chain rotating downward towards the heme, furtherclosing off the entrance to the pocket at the top distal side of thesubdomain (FIG. 3B). In this position it appears that the NE atom ofArg-114 makes a long-range hydrogen bond (3.94 Å) with the O1D oxygenatom of the ‘D’ ring carboxylate.

The basis of interspecies variations in albumin heme binding is furtherindicated by the high resolution structure of the present invention. Asdiscussed above, there appear to be five residues which show strong or‘key’ interactions with the heme: Tyr 161, Ile-142, Tyr-138, His 146,and Lys-190. In mammals all of these residues are either conserved(Tyr-161, Tyr-138, His-146) or substituted with closely homologousresidues, i.e., Ile-142 to Val. with the important exception of Lys-190.Lys-190, the ‘gate’ residue, while present in primate albumins, isreplaced with Leu in all of the currently sequenced mammalian albumins.This strongly suggests that the salt bridging interaction of Lys-190 iskey to stabilizing and securing the heme within the binding pocket. Theimportance of this residue is further corroborated by the sequence ofbull frog albumin (27), which has retained Lys at 190, and despite itsoverall low sequence identity with mammalian albumins (21.7%), stillretains its heme binding capabilities. The occurrence of Lys, His or Argat 190 (normalized to the amino acid sequence of human serum albumin),allows the prediction that several other albumins such as hen (28) andthe other members of the albumin gene family e.g., alpha-fetoprotein(29) will also be found to harbor an active high affinity heme bindingsite. Lys-190 may also play an important role the initial ‘fast’ bindinginteraction proposed by Adams and Berman (9) followed by the slowerinternalization of the heme into the hydrophobic pocket. Finally, itshould also be considered that the presence of Leu at the pocketopening, may, in addition to eliminating an important chargestabilization, sterically hinder heme access to the pocket.

It is therefore another aspect of the present invention that impartingheme-binding properties into albumins of non-human origin may beachieved by replacing the inactivating equivalent residue (equivalent toresidue 190 in human serum albumin) with Lys or His. In many non-humanalbumins, particularly mammalian albumins, the amino acid residue at theequivalent position of the human aa 190 (as determined by sequencealignment of albumins well known to those skilled in the art and shown,e.g., in Carter et al., (1994), The Structure of Serum Albumin. Adv.Prot. Chem., 45, 153-203, incorporated herein by reference) has a Leuresidue instead of Lys. As a result, these albumins do not normally bindto heme, and thus the substitution of Lys for Leu in these non-humanalbumins at the equivalent position of HSA Lys-190 would result in theimparting of heme-binding properties to non-human albumins, particularlynon-human mammalian albumins.

In accordance with the present invention, a method is provided fordeveloping molecules of albumin with improved gas binding propertiesusing the crystal high resolution structure described above. It has longbeen established that the native hemalbumin complex is inactive as anoxygen binding protein, a property that is now readily understood by thechemistry of the observed structure. Since albumin is the major proteinof the circulatory system contributing 80% to osmotic blood pressure andmaintenance of blood pH (4) and considered to be the volume expander ofchoice; it is believed that the development of an oxygen transportingalbumin could be of tremendous medical importance. For example, others,in a different approach from the present invention, have produced amodified heme with an axial imidazole base covalently linked to theporphyrin ring (30) which has been shown to reversibly bind oxygen invitro and in vivo (16, 31-35), although the physical size of FePprobably precludes it from binding to the same high affinity site ashemin.

The atomic structure of methemalbumin as described above provides anessential framework for the development of albumin-based ‘bloodsubstitutes’ or improved volume expanders with oxygen transportcapabilities. The high resolution hemalbumin structures of the inventionhave allowed for the determination of the key regions which will allowfor the creation of genetically engineered albumins with novel gasbinding properties. For example, substitution of the selected residuesin closest proximity to the heme with histidine, activates the heme tolimited reversible oxygen binding in the Fell state (19). As set forthabove, these key residues include amino acids Tyr-161, Phe-157, Arg-186,Leu-182, Arg-117, Phe-134, Leu-135, Leu-154, Phe-149, Ile-142, His-146,Arg-114, Lys-190, Ser-193, Ala-158, Tyr-138, Leu-115, Met-123, Phe-165,and Pro-118 of serum albumin.

In accordance with the invention, the provision of the high resolutionatomic coordinates of the hemalbumin complex will also allow for theidentification of key binding sites such as to permit the development ofgenetically modified albumin and heme molecules which have maximized gasbinding properties. This can be done by examining the high resolutionstructure with regard to the interface with gases such as oxygen andother potentially toxic gases such as carbon monoxide, nitric acid andcyanide, and by maximizing the ability of the complex to bind with suchgases by replacement of residues as necessary to maximize the binding tospecific gases. Accordingly, another aspect of the invention will be theimprovement of binding, transport, and delivery of therapeutic gasessuch as oxygen, as well as the scavenging and removal of potentiallytoxic gases such as cyanide, nitric acid, carbon monoxide, etc.

It is thus submitted that the foregoing embodiments are onlyillustrative of the claimed invention, and alternative embodiments wellknown or obvious to one skilled in the art not specifically set forthabove also fall within the scope of the claims.

In addition, the following examples are presented as illustrative of theclaimed invention, or aspects associated with the present invention, andare not deemed to be limiting of the scope of the invention in anymanner.

EXAMPLES Example 1 Obtaining the High Resolution Structure of theHemalbumin Complex

Overview:

The high resolution structure of hemalbumin was determined by singlecrystal X-ray diffraction to a resolution of 1.9 Å. The structurerevealed the protoporphyrin IX bound to a single site within ahydrophobic cavity in subdomain IB, one of the principal binding sitesfor long chain fatty acid. The iron is penta coordinated with the fifthligand comprised of the hydroxyloxygen of Tyr-161 (phenolic oxygen toheme plane distance: 2.73 Å) in an otherwise completely hydrophobicpocket. The heme propionic acid residues form salt bridges with His-142and Lys-190, which together with a series of hydrophobic interactions,enclose and secure the heme within the IB helical motif. A detaileddiscussion of the structure together with its implications for thedevelopment of potential blood substitutes is presented.

Materials and Methods

The hemalbumin crystals were prepared in a manner previously described(19,20), with the following exception, the human albumin, defatted afterChen (21) was coupled with 1:1 molar ratio of albumin:hemin(Fe^(III)-Protoporphyrin-IX (Cl)) prior to coupling with myristate. Thecrystals are of the same C2 form as the form-III of our previous report(19). Diffraction data were collected at the Beamline 5.0.3 of theAdvanced Light Source (ALS), Lawrence Berkeley Laboratory (LBL). A 2×2array (ADSC) of CCD detectors was used with a detector-to-crystaldistance of 180 mm. A total of 180 (1°) oscillation images werecollected from one crystal at 100 K using photon wavelength of 1.0 Å andexposure of 60 second per frame. At the cryogenic temperature, the unitcell dimensions were a=183.11 Å, b=37.91 Å, c=94.83 Å, β=105.04°. Theseimages yielded a data set of 44,335 independent reflections withpositive intensities to a resolution of 1.9 Å (R_(merge): 3.7%; averagemultiplicity: 2.03; average l/σ(1): 14.7, and completeness: 88.1%).

The program package of CNX X-ray from Accelrys, Inc. was used forstructure refinement, with the protein starting model taken from astructure of human albumin complexed with myristate (without hemin)determined in our earlier studies (19,22) and re-refined more recentlyat 100 K (unpublished). The hemin and five myristate molecules wereclearly resolved in the difference density maps after initialrefinement. When hemin, myristates, and solvent water molecules wereincorporated in the model, the refinement quickly converged to anR-factor of 22.8% for all 42,107 reflections in the working set with nosigma cut-off (R-free 28.2%), yielding a structure of good geometrygiving rms deviations from ideality of 0.005 Å for bond-distance and1.190 for valence-angle (Table I). The final model includes 583 proteinresidues, 1 hemin molecule, 5 myristic acid molecules and 581 solventwater molecules with an average B-factor of 27.8 Å² for the proteinatoms. One residue each at both N- and C-termini were not visible on theelectron density maps, presumably disordered, and were not included inthe model. Atomic coordinates of the methemalbumin structure areprovided herein in Appendix A. TABLE I Data collection and modelrefinement statistics Data collection Space group C2 a (Å) 183.116 b (Å)37.909 c (Å) 94.832 β (°) 105.036 Resolution range (Å) 50-1.9(1.97-1.9)^(a) Independent reflections 45,420 Multiplicity 2.6Completeness (%) 83.5 (56.3)^(a) I/_(σI) ^(b) 14.7 R_(sym) (%)^(c) 3.7(26.6)^(a) Number of crystals used 1 Temperature of data collection (K)100 X-ray wavelength (Å) 1.0 Model refinement Number of non-hydrogenatoms 4,759 Number of water molecules 581 R_(work) (%)^(d) 22.8 R_(free)(%)^(e) 28.2 rms^(f) deviation from ideal bond lengths (Å) 0.005 rmsdeviation from ideal bond angles (°) 1.19 rms deviation in B-factorsmain/side chain (Å) 1.382/2.327 Average B-factor (Å²) 27.8^(a)Values in parentheses indicate the highest resolution shell.^(b)NO I/_(σI) cutoff was used in the refinement.^(c)R_(sym) = Σ|(I_(hkl)) − <I>|/Σ(I_(hkl)), where I_(hkl) is theintegrated intensity of a given reflection.^(d)R_(work) = Σ_(hkl)|F_(obs) − F_(calc)|/Σ_(hkl)F_(obs) where F_(obs)and F_(calc) are the observed and calculated structure factors,respectively (identical to R_(cryst)).^(e)R_(free) is the R_(work) calculated using a randomly selected 5%sample of reflection data omitted from the refinement.^(f)rms means root mean square.Results and Discussion

The current hemalbumin structure is well determined and represents thehighest resolution albumin structure reported to date. Details of therefinement statistics have been given in Table I. The quality of theresulting electron density is high providing detailed information of theheme binding interaction as well as other important structuralinformation such as positions of key water molecules. The orientation ofthe heme (Fe^(III)-Protoporphyrin-IX) provides a good fit to theelectron density as shown in FIG. 1. There is the possibility that amixture of the two orientations actually exists in the complex (180degree rotational isomers along the CHA to CHC line). However, theorientation chosen provides the best fit, and neither orientationaffects the resulting interpretation of the binding chemistry. Theoverall structure of methemalbumin is very similar to the HSA-myristatestructure we have previously determined and used in this work to derivethe phases for the methemalbumin structure (discussed above); and thestructure using the same crystal forms and reported by others (23). Thestructures are essentially identical, having a rms deviation between Caatom of positions of 0.396 Å. When Cα for residues 109-195 thatconstitute the IB subdomain are superimposed, the rms was 0.32 Å.

A single site for hemin revealed by earlier lower resolution studies(19) was verified by this high resolution study. The hemin is locatedwithin the IB subdomain which is constituted by a loop and fourcontiguous helices (h7, h8, h9 and h10) (22,24). The heme is buried in ahydrophobic cleft or pocket formed by the subdomain helices and itsposition within IB forms a striking superposition on the planerepresented by the curved structure assumed by long chain fatty acidwhen occupying this site (FIG. 2). One half of the pocket is enclosed byh9 and h10, the other half by the loop h8, the top by h7 and the bottomby the loop between h8 and h9. The plane of the inserted heme liesapproximately 30 degrees to the helical axes of h9 and h10.

There are five residues that show close interaction with the heme andappear to be key contributors to the high binding affinity, Tyr-161,Ile-142, Tyr-138, His-145, and Lys-190. The tyrosine side chains formclose parallel stacking interactions with the heme (FIG. 3). The mostinteresting heme interaction involves Tyr-161, which shows coordinationof the phenolic oxygen with the heme iron resulting in an oxygen to hemeplane distance of 2.73 Å and a pentacoordinate iron. This phenolicoxygen replaces the chloride ion of the hemin complex in solution(Fe^(III)-Protoporphyrin-IX (Cl)). The details of the methemalbumin hemeare similar to the original hemin small molecule structure determined in1965 (25) where: 1) the iron is located off the heme surface (0.475 Å)(the Fe electron bulge in the methemalbumin indicates a similarmovement, see FIG. 1); 2) the Chloride to heme plane distance is 2.693 Å(methemalbumin Tyr-161 O to heme plane distance 2.73 Å); and 3) hemepotentially disordered (averaged) by the two hemes rotated 180° about aline between the CHA and CHC atoms in both structures. In themethemalbumin structure the Fe atom was restricted to co-planarity withthe heme during refinement, however, the difference maps as in FIG. 1,show a significant deviation from planarity, suggesting an iron positionsimilar to the original hemin small molecule structure. If one assumesthe iron atom also lies 0.475 Å off the heme surface in methemalbumin,then the Tyr-161 phenyl oxygen to iron distance can be estimated at 2.27Å, a value in good agreement with the Cl to Fe distance of 2.22 Å inhemin (25). In the more recent malarial pigment β-hematin structurewhere the carboxylate oxygen of a symmetrically related heme becomes thefifth coordinate to the Fe atom, the O to Fe distance was determined at1.89 Å (26).

In addition to the coordination with the heme iron, the phenolic oxygenof Tyr-161 forms hydrogen bonds with a series of water molecules (W1058,W1099, and W1244) within the hydrophobic cavity and extending into awell defined surface water structure (FIG. 3B). We shall, for the sakeof this example, refer to this side of the heme as the “proximal” side.No water molecules were observed on the opposite or ‘distal’ side of theheme pocket. The propionic residues of the heme protrude from the pocketand form two important salt bridges, one with His-145 which forms astrong bridge with the ‘A’ ring carboxylate and the other with Lys-190,which forms a salt bridge mid way between both the ‘A’ and ‘D’ ring hemecarboxyls (FIG. 3). Ile-142, near the iron on the distal side of theheme, contributes one of the closest hydrophobic interactions.

A list of the residues having close interaction or contributing to thehydrophobic surface of the binding pocket are: Tyr-161, Phe-157,Arg-186, Leu-182, Arg-117, Phe-134, Leu-135, Leu-154, Phe-149, Ile-142,His-146, Arg-114, Lys-190, Ser-193, Ala-158, Tyr-138, Leu-115, Met-123,Phe-165, and Pro-118. Details of many of the heme binding interactionsof these residues are illustrated in FIG. 3.

Comparisons of the hemalbumin structure with the nativealbumin/myristate structure reveal four residues that show pronouncedmovements upon heme complexation. The first of these is Ile-142 wherethe CD1 atom has swung about 135° away from the heme plane to avoidsteric clash, resulting in a CD1 atom within 3.38 Å from the NAnitrogen. His-146 displays perhaps the largest movement uponcomplexation, making a key salt bridge with the ‘A’ ring carboxylate ofheme, resulting in an equidistant His NE atom to O1A/O2A distance of3.30 Å. The third residue demonstrating significant side chain movementis Lys-190. In the HSA-myristate structure, the NZ nitrogen at the endof the side chain of Lys-190 makes a 2.99 Å salt bridge with the OD1oxygen of Asp-187. In the methemalbumin structure, this salt bridge isbroken resulting in the 180° rotation of the Lys-190 side chain aboutits CD atom across the pocket opening to form a key salt bridgeinteraction with both the heme carboxyls. This new position wassupported by clear difference electron density. In this position, the NZnitrogen atom of Lys-190 is almost midway between the two hemecarboxylate groups and makes a salt bridge with each of them. Theshortest (2.99 Å) of these is made with the O2D oxygen of the heme ‘D’ring carboxylate, while a slightly longer salt bridge (3.32 Å) is formedbetween the NZ atom of Lys-190 and the O1A oxygen of the heme ‘A’ ringcarboxylate. The swinging motion by the side chain of Lys-190 can belikened to a gate shutting after the hemin/heme is bound with the saltbridges serving as a latch to keep the gate shut. Arg-114, the fourthresidue, has its side chain rotating downward towards the heme, furtherclosing off the entrance to the pocket at the top distal side of thesubdomain (FIG. 3B). In this position it appears that the NE atom ofArg-114 makes a long-range hydrogen bond (3.94 Å) with the O1D oxygenatom of the ‘D’ ring carboxylate.

Structural Basis of Interspecies Differences in Hemin Binding

The basis of interspecies variations in albumin heme binding is clearlyindicated by this structure. As discussed in the previous section, thereare essentially five residues, which show strong or ‘key’ interactionswith the heme: Tyr 161, Ile-142, Tyr-138, His 146, and Lys-190. Inmammals all of these residues are either conserved (Tyr-161, Tyr-138,His-146) or substituted with closely homologous residues, i.e., Ile-142to Val. with the important exception of Lys-190. Lys-190, the ‘gate’residue, while present in primate albumins, is replaced with Leu in allof the currently sequenced mammalian albumins. This strongly suggeststhat the salt bridging interaction of Lys-190 is key to stabilizing andsecuring the heme within the binding pocket. The importance of thisresidue is further corroborated by the sequence of bull frog albumin(27), which has retained Lys at 190, and despite its overall lowsequence identity with mammalian albumins (21.7%), still retains itsheme binding capabilities. The occurrence of Lys, His or Arg at 190(normalized to the amino acid sequence of human serum albumin), allowsthe prediction that several other albumins such as hen (28) and theother members of the albumin gene family e.g., alpha-fetoprotein (29)will also be found to harbor an active high affinity heme binding site.It may be further the case that Lys-190 plays an important role theinitial ‘fast’ binding interaction proposed by Adams and Berman (9)followed by the slower internalization of the heme into the hydrophobicpocket. Finally, it should also be considered that the presence of Leuat the pocket opening, may, in addition to eliminating an importantcharge stabilization, sterically hinder heme access to the pocket.

Implications For the Creation of Albumins with Novel Gas BindingProperties

It has long been established that the native hemalbumin complex isinactive as an oxygen binding protein, a property that is now readilyunderstood by the chemistry of the observed structure. Since albumin isthe major protein of the circulatory system contributing 80% to osmoticblood pressure and maintenance of blood pH (4) and considered to be thevolume expander of choice; it is believed that the further developmentand refinement of an oxygen transporting albumin (such as disclosed inU.S. Pat. No. 5,948,609) could be of tremendous medical importance.

For example, others, in a different approach from the present invention,have produced a modified heme with an axial imidazole base covalentlylinked to the porphyrin ring (30) which has been shown to reversiblybind oxygen in vitro and in vivo (16, 31-35), although the physical sizeof FeP probably precludes it from binding to the same high affinity siteas hemin. The atomic structure of methemalbumin in accordance with theinvention thus provides an essential framework for the development ofalbumin-based ‘blood substitutes’ or improved volume expanders withoxygen transport capabilities. The hemalbumin complexes havesuccessfully guided the creation of genetically engineered albumins withnovel gas binding properties. For example, simple substitution of theselected residues in closest proximity to the heme with histidine,activates the heme to limited reversible oxygen binding in the Fellstate (19) and/or imparts high affinity to CN in the oxidized Fe^(III)form.

In general, the high resolution structure revealed numerous previouslyunappreciated components of the interaction of albumin withprotoporphyrins. For example we now understand the species variation inheme binding or lack thereof.

We also understand which side of the pocket provides the distal andproximal interactions to the heme, the role and accessible surface towater molecules, the residues important in creating the high affinity tothe heme and many many other exacting points relevant to therapeuticdevelopment.

Accordingly, It should be understood by one skilled in the art that thisinformation may now make possible various ab initio calculations todesign and improve heme or oxygen binding or any other methods oftherapeutic development (since these are now the highest resolutionhuman albumin coordinates). The present invention can thus be used toguide rational drug design of therapeutic compounds associated with thehemalbumin complex, in terms of drugs that can inhibit or enhancecertain functions, or in terms of products such as recombinant forms ofhemalbumin which can be constructed to reversibly bind oxygen.

REFERENCES

The following citations are incorporated by reference as if set forth infull herein:

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APPENDIX A

List of atomic coordinates of subdomain IB of human albumin and heme.

Note:

-   -   (1) Atomic coordinates list is part of the ternary complex        crystal structure of human albumin, heme and myristate;    -   (2) Crystal form is of space group of C2 (with b as unique axis)        with cell dimensions of a=183.1 Å, b=37.9 Å, c=94.8 Å, (=105.0        (;    -   (3) The orthogonal coordinates span in the Cartesian coordinate        system which conforms to the same convention as that of the        Protein Data Bank;

(4) List also includes selected solvent water molecules Involved ininteractions with heme. ATOM 852 N ASN 109 34.561 −7.463 27.701 1.0037.28 N ATOM 853 CA ASN 109 33.707 −7.643 28.877 1.00 38.81 C ATOM 854CB ASN 109 32.588 −8.649 28.585 1.00 41.49 C ATOM 855 CG ASN 109 31.563−8.724 29.708 1.00 44.62 C ATOM 856 OD1 ASN 109 31.914 −8.880 30.8771.00 47.83 O ATOM 857 ND2 ASN 109 30.288 −8.621 29.354 1.00 46.00 N ATOM858 C ASN 109 34.612 −8.186 29.979 1.00 37.18 C ATOM 859 O ASN 10934.498 −9.345 30.362 1.00 38.56 O ATOM 860 N PRO 110 35.529 −7.34630.491 1.00 35.52 N ATOM 861 CD PRO 110 35.650 −5.926 30.108 1.00 34.37C ATOM 862 CA PRO 110 36.495 −7.677 31.546 1.00 35.82 C ATOM 863 CB PRO110 37.407 −6.452 31.564 1.00 34.23 C ATOM 864 CG PRO 110 36.464 −5.34731.256 1.00 35.17 C ATOM 865 C PRO 110 35.915 −7.988 32.927 1.00 36.43 CATOM 866 O PRO 110 36.652 −8.235 33.877 1.00 38.59 O ATOM 867 N ASN 11134.591 −7.979 33.020 1.00 36.82 N ATOM 868 CA ASN 111 33.897 −8.25734.273 1.00 37.30 C ATOM 869 CB ASN 111 33.908 −9.763 34.550 1.00 39.93C ATOM 870 CG ASN 111 33.026 −10.538 33.583 1.00 42.45 C ATOM 871 OD1ASN 111 31.831 −10.255 33.458 1.00 42.90 O ATOM 872 ND2 ASN 111 33.611−11.507 32.890 1.00 44.99 N ATOM 873 C ASN 111 34.432 −7.483 35.472 1.0036.36 C ATOM 874 O ASN 111 34.707 −8.044 36.538 1.00 38.31 O ATOM 875 NLEU 112 34.580 −6.179 35.291 1.00 33.54 N ATOM 876 CA LEU 112 35.027−5.300 36.351 1.00 30.14 C ATOM 877 CB LEU 112 35.866 −4.147 35.774 1.0031.52 C ATOM 878 CG LEU 112 37.256 −4.418 35.198 1.00 32.24 C ATOM 879CD1 LEU 112 37.696 −3.245 34.311 1.00 32.84 C ATOM 880 CD2 LEU 11238.239 −4.610 36.337 1.00 33.20 C ATOM 881 C LEU 112 33.761 −4.71936.988 1.00 27.71 C ATOM 882 O LEU 112 32.703 −4.687 36.347 1.00 25.69 OATOM 883 N PRO 113 33.839 −4.262 38.256 1.00 27.01 N ATOM 884 CD PRO 11334.990 −4.290 39.179 1.00 26.45 C ATOM 885 CA PRO 113 32.650 −3.68438.906 1.00 27.36 C ATOM 886 CB PRO 113 33.163 −3.283 40.290 1.00 26.92C ATOM 887 CG PRO 113 34.312 −4.241 40.538 1.00 30.79 C ATOM 888 C PRO113 32.207 −2.469 38.100 1.00 26.20 C ATOM 889 O PRO 113 33.029 −1.75437.536 1.00 27.92 O ATOM 890 N ARG 114 30.906 −2.233 38.055 1.00 26.82 NATOM 891 CA ARG 114 30.386 −1.103 37.303 1.00 30.05 C ATOM 892 CB ARG114 28.870 −1.243 37.160 1.00 32.33 C ATOM 893 CG ARG 114 28.323 −0.81535.809 1.00 40.32 C ATOM 894 CD ARG 114 26.891 −1.299 35.582 1.00 44.41C ATOM 895 NE ARG 114 25.976 −0.804 36.602 1.00 49.10 N ATOM 896 CZ ARG114 24.659 −0.972 36.557 1.00 51.47 C ATOM 897 NH1 ARG 114 23.885 −0.48837.524 1.00 53.34 N ATOM 898 NH2 ARG 114 24.114 −1.632 35.545 1.00 53.41N ATOM 899 C ARG 114 30.746 0.178 38.056 1.00 29.43 C ATOM 900 O ARG 11430.750 0.198 39.290 1.00 26.16 O ATOM 901 N LEU 115 31.071 1.239 37.3221.00 27.53 N ATOM 902 CA LEU 115 31.415 2.508 37.942 1.00 26.31 C ATOM903 CB LEU 115 31.997 3.484 36.921 1.00 24.86 C ATOM 904 CG LEU 11533.355 3.144 36.308 1.00 26.44 C ATOM 905 CD1 LEU 115 33.785 4.25835.361 1.00 25.22 C ATOM 906 CD2 LEU 115 34.385 2.959 37.413 1.00 27.00C ATOM 907 C LEU 115 30.164 3.118 38.542 1.00 26.77 C ATOM 908 O LEU 11529.091 3.061 37.949 1.00 27 .27 O ATOM 909 N VAL 116 30.309 3.697 39.7261.00 27.29 N ATOM 910 CA VAL 116 29.182 4.333 40.383 1.00 28.84 C ATOM911 CB VAL 116 28.845 3.642 41.721 1.00 31.05 C ATOM 912 CG1 VAL 11628.583 2.150 41.488 1.00 33.07 C ATOM 913 CG2 VAL 116 29.970 3.85842.721 1.00 31.47 C ATOM 914 C VAL 116 29.494 5.803 40.639 1.00 28.00 CATOM 915 O VAL 116 30.611 6.171 40.996 1.00 27.97 O ATOM 916 N ARG 11728.491 6.641 40.452 1.00 28.04 N ATOM 917 CA ARG 117 28.650 8.070 40.6571.00 27.31 C ATOM 918 CB ARG 117 27.482 8.800 40.002 1.00 27.89 C ATOM919 CG ARG 117 27.532 10.302 40.108 1.00 26.30 C ATOM 920 CD ARG 11726.247 10.892 39.561 1.00 27.94 C ATOM 921 NE ARG 117 26.047 10.57338.150 1.00 29.94 N ATOM 922 CZ ARG 117 24.988 10.950 37.439 1.00 30.00C ATOM 923 NH1 ARG 117 24.898 10.616 36.162 1.00 28.86 N ATOM 924 NH2ARG 117 24.015 11.656 38.006 1.00 29.25 N ATOM 925 C ARG 117 28.7248.412 42.144 1.00 27.03 C ATOM 926 O ARG 117 27.794 8.138 42.903 1.0025.34 O ATOM 927 N PRO 118 29.847 9.005 42.584 1.00 27.40 N ATOM 928 CDPRO 118 31.093 9.250 41.841 1.00 27.56 C ATOM 929 CA PRO 118 30.0019.372 43.995 1.00 27.93 C ATOM 930 CB PRO 118 31.465 9.805 44.091 1.0028.17 C ATOM 931 CG PRO 118 32.123 9.101 42.920 1.00 29.07 C ATOM 932 CPRO 118 29.071 10.527 44.310 1.00 28.14 C ATOM 933 O PRO 118 28.43411.083 43.411 1.00 27.59 O ATOM 934 N GLU 119 28.984 10.883 45.586 1.0028.20 N ATOM 935 CA GLU 119 28.155 12.007 45.970 1.00 29.04 C ATOM 936CB GLU 119 28.028 12.090 47.491 1.00 31.35 C ATOM 937 CG GLU 119 27.07111.066 48.078 1.00 34.74 C ATOM 938 CD GLU 119 25.721 11.093 47.390 1.0037.65 C ATOM 939 OE1 GLU 119 25.511 10.298 46.446 1.00 39.19 O ATOM 940OE2 GLU 119 24.874 11.926 47.780 1.00 38.84 O ATOM 941 C GLU 119 28.85113.250 45.428 1.00 27.64 C ATOM 942 O GLU 119 30.077 13.288 45.326 1.0025.66 O ATOM 943 N VAL 120 28.062 14.257 45.073 1.00 27.22 N ATOM 944 CAVAL 120 28.588 15.502 44.525 1.00 26.74 C ATOM 945 CB VAL 120 27.45416.533 44.357 1.00 26.55 C ATOM 946 CG1 VAL 120 28.018 17.882 43.9311.00 25.02 C ATOM 947 CG2 VAL 120 26.455 16.021 43.339 1.00 27.22 C ATOM948 C VAL 120 29.700 16.130 45.366 1.00 26.28 C ATOM 949 O VAL 12030.726 16.557 44.831 1.00 24.35 O ATOM 950 N ASP 121 29.491 16.20246.679 1.00 24.10 N ATOM 951 CA ASP 121 30.488 16.796 47.563 1.00 26.11C ATOM 952 CB ASP 121 29.989 16.788 49.011 1.00 32.56 C ATOM 953 CG ASP121 28.756 17.648 49.209 1.00 37.62 C ATOM 954 OD1 ASP 121 28.013 17.41150.189 1.00 42.25 O ATOM 955 OD2 ASP 121 28.531 18.568 48.392 1.00 41.57O ATOM 956 C ASP 121 31.801 16.030 47.461 1.00 25.21 C ATOM 957 O ASP121 32.884 16.627 47.414 1.00 24.52 O ATOM 958 N VAL 122 31.700 14.70547.424 1.00 22.72 N ATOM 959 CA VAL 122 32.876 13.859 47.326 1.00 23.61C ATOM 960 CB VAL 122 32.501 12.365 47.473 1.00 23.21 C ATOM 961 CG1 VAL122 33.729 11.499 47.241 1.00 22.69 C ATOM 962 CG2 VAL 122 31.929 12.10648.862 1.00 24.86 C ATOM 963 C VAL 122 33.599 14.060 45.995 1.00 23.75 CATOM 964 O VAL 122 34.817 14.229 45.962 1.00 24.33 O ATOM 965 N MET 12332.847 14.036 44.901 1.00 25.05 N ATOM 966 CA MET 123 33.426 14.21243.571 1.00 25.29 C ATOM 967 CB MET 123 32.338 14.206 42.503 1.00 25.78C ATOM 968 CG MET 123 31.662 12.894 42.275 1.00 27.47 C ATOM 969 SD MET123 30.635 13.039 40.826 1.00 25.52 S ATOM 970 CE MET 123 29.038 13.40841.522 1.00 25.19 C ATOM 971 C MET 123 34.153 15.540 43.472 1.00 25.03 CATOM 972 O MET 123 35.328 15.606 43.118 1.00 22.07 O ATOM 973 N CYS 12433.417 16.603 43.769 1.00 25.57 N ATOM 974 CA CYS 124 33.957 17.94743.709 1.00 25.21 C ATOM 975 C CYS 124 35.196 18.147 44.547 1.00 25.46 CATOM 976 O CYS 124 36.124 18.850 44.142 1.00 25.74 O ATOM 977 CB CYS 12432.890 18.943 44.131 1.00 24.57 C ATOM 978 SG CYS 124 31.720 19.27942.796 1.00 26.71 S ATOM 979 N THR 125 35.207 17.536 45.724 1.00 25.24 NATOM 980 CA THR 125 36.343 17.653 46.614 1.00 24.06 C ATOM 981 CB THR125 35.998 17.118 48.009 1.00 25.11 C ATOM 982 OG1 THR 125 34.961 17.92748.574 1.00 25.31 O ATOM 983 CG2 THR 125 37.209 17.155 48.917 1.00 26.39C ATOM 984 C THR 125 37.536 16.897 46.051 1.00 24.49 C ATOM 985 O THR125 38.669 17.367 46.128 1.00 24.80 O ATOM 986 N ALA 126 37.279 15.72845.475 1.00 23.51 N ATOM 987 CA ALA 126 38.353 14.937 44.901 1.00 23.39C ATOM 988 CB ALA 126 37.825 13.576 44.452 1.00 25.21 C ATOM 989 C ALA126 38.895 15.719 43.713 1.00 23.10 C ATOM 990 O ALA 126 40.104 15.81843.526 1.00 22.38 O ATOM 991 N PHE 127 37.982 16.281 42.925 1.00 23.88 NATOM 992 CA PHE 127 38.336 17.074 41.757 1.00 26.00 C ATOM 993 CB PHE127 37.063 17.649 41.118 1.00 26.05 C ATOM 994 CG PHE 127 37.319 18.51139.915 1.00 28.05 C ATOM 995 CD1 PHE 127 37.759 17.951 38.718 1.00 29.22C ATOM 996 CD2 PHE 127 37.142 19.888 39.984 1.00 28.78 C ATOM 997 CE1PHE 127 38.020 18.752 37.607 1.00 27.59 C ATOM 998 CE2 PHE 127 37.40020.700 38.881 1.00 29.70 C ATOM 999 CZ PHE 127 37.841 20.128 37.688 1.0029.75 C ATOM 1000 C PHE 127 39.270 18.205 42.178 1.00 27.81 C ATOM 1001O PHE 127 40.319 18.426 41.567 1.00 25.51 O ATOM 1002 N HIS 128 38.88518.911 43.237 1.00 28.91 N ATOM 1003 CA HIS 128 39.675 20.026 43.7521.00 30.38 C ATOM 1004 CB HIS 128 38.920 20.740 44.877 1.00 32.39 C ATOM1005 CG HIS 128 39.721 21.811 45.549 1.00 35.91 C ATOM 1006 CD2 HIS 12840.401 21.813 46.720 1.00 37.04 C ATOM 1007 ND1 HIS 128 39.936 23.04944.980 1.00 37.93 N ATOM 1008 CE1 HIS 128 40.714 23.766 45.771 1.0037.78 C ATOM 1009 NE2 HIS 128 41.010 23.039 46.834 1.00 36.51 N ATOM1010 C HIS 128 41.040 19.618 44.280 1.00 30.75 C ATOM 1011 O HIS 12842.036 20.294 44.028 1.00 30.33 O ATOM 1012 N ASP 129 41.090 18.52345.030 1.00 31.65 N ATOM 1013 CA ASP 129 42.356 18.075 45.596 1.00 33.00C ATOM 1014 CB ASP 129 42.130 16.891 46.544 1.00 34.16 C ATOM 1015 CGASP 129 41.220 17.242 47.710 1.00 36.93 C ATOM 1016 OD1 ASP 129 41.16218.433 48.088 1.00 37.00 O ATOM 1017 OD2 ASP 129 40.573 16.324 48.2581.00 38.07 O ATOM 1018 C ASP 129 43.380 17.701 44.529 1.00 33.92 C ATOM1019 O ASP 129 44.585 17.862 44.730 1.00 33.33 O ATOM 1020 N ASN 13042.903 17.199 43.394 1.00 34.53 N ATOM 1021 CA ASN 130 43.803 16.81942.312 1.00 34.04 C ATOM 1022 CB ASN 130 44.645 15.612 42.728 1.00 36.13C ATOM 1023 CG ASN 130 45.770 15.316 41.746 1.00 39.82 C ATOM 1024 OD1ASN 130 46.644 14.497 42.022 1.00 43.50 O ATOM 1025 ND2 ASN 130 45.74815.978 40.594 1.00 38.81 N ATOM 1026 C ASN 130 43.024 16.503 41.045 1.0032.63 C ATOM 1027 O ASN 130 42.692 15.348 40.778 1.00 31.11 O ATOM 1028N GLU 131 42.739 17.546 40.275 1.00 30.08 N ATOM 1029 CA GLU 131 41.99717.424 39.026 1.00 32.21 C ATOM 1030 CB GLU 131 41.879 18.790 38.3461.00 33.42 C ATOM 1031 CG GLU 131 40.899 19.737 39.001 1.00 39.40 C ATOM1032 CD GLU 131 40.958 21.134 38.412 1.00 41.82 C ATOM 1033 OE1 GLU 13141.060 21.256 37.171 1.00 42.45 O ATOM 1034 OE2 GLU 131 40.890 22.10839.195 1.00 43.51 O ATOM 1035 C GLU 131 42.647 16.455 38.051 1.00 30.48C ATOM 1036 O GLU 131 41.965 15.652 37.415 1.00 28.19 O ATOM 1037 N GLU132 43.966 16.552 37.928 1.00 30.36 N ATOM 1038 CA GLU 132 44.720 15.70237.013 1.00 30.68 C ATOM 1039 CB GLU 132 46.219 15.971 37.157 1.00 34.57C ATOM 1040 CG GLU 132 46.612 17.440 37.283 1.00 41.76 C ATOM 1041 CDGLU 132 46.367 18.243 36.018 1.00 45.66 C ATOM 1042 OE1 GLU 132 46.93519.351 35.910 1.00 48.52 O ATOM 1043 OE2 GLU 132 45.609 17.776 35.1381.00 48.47 O ATOM 1044 C GLU 132 44.460 14.230 37.289 1.00 28.06 C ATOM1045 O GLU 132 43.965 13.502 36.430 1.00 27.02 O ATOM 1046 N THR 13344.807 13.798 38.496 1.00 25.26 N ATOM 1047 CA THR 133 44.635 12.40838.903 1.00 23.70 C ATOM 1048 CB THR 133 45.224 12.176 40.313 1.00 25.41C ATOM 1049 OG1 THR 133 46.634 12.426 40.277 1.00 27.80 O ATOM 1050 CG2THR 133 44.973 10.741 40.785 1.00 24.34 C ATOM 1051 C THR 133 43.17611.974 38.895 1.00 23.48 C ATOM 1052 O THR 133 42.854 10.867 38.470 1.0022.17 O ATOM 1053 N PHE 134 42.300 12.851 39.372 1.00 21.62 N ATOM 1054CA PHE 134 40.874 12.568 39.420 1.00 22.13 C ATOM 1055 CB PHE 134 40.12813.788 39.966 1.00 22.53 C ATOM 1056 CG PHE 134 38.638 13.625 40.0001.00 22.38 C ATOM 1057 CD1 PHE 134 38.039 12.760 40.908 1.00 22.96 CATOM 1058 CD2 PHE 134 37.834 14.325 39.113 1.00 21.91 C ATOM 1059 CE1PHE 134 36.661 12.598 40.929 1.00 22.17 C ATOM 1060 CE2 PHE 134 36.45914.171 39.124 1.00 20.58 C ATOM 1061 CZ PHE 134 35.868 13.307 40.0341.00 22.88 C ATOM 1062 C PHE 134 40.363 12.232 38.018 1.00 21.41 C ATOM1063 O PHE 134 39.693 11.216 37.813 1.00 22.84 O ATOM 1064 N LEU 13540.690 13.101 37.068 1.00 20.12 N ATOM 1065 CA LEU 135 40.292 12.94535.672 1.00 22.65 C ATOM 1066 CB LEU 135 40.600 14.231 34.899 1.00 23.79C ATOM 1067 CG LEU 135 39.478 15.225 34.575 1.00 26.03 C ATOM 1068 CD1LEU 135 38.372 15.137 35.586 1.00 25.72 C ATOM 1069 CD2 LEU 135 40.06816.633 34.504 1.00 23.26 C ATOM 1070 C LEU 135 40.981 11.767 34.990 1.0021.48 C ATOM 1071 O LEU 135 40.337 10.976 34.307 1.00 20.90 O ATOM 1072N LYS 136 42.292 11.652 35.161 1.00 22.49 N ATOM 1073 CA LYS 136 43.00710.554 34.530 1.00 21.84 C ATOM 1074 CB LYS 136 44.519 10.740 34.6791.00 24.08 C ATOM 1075 CG LYS 136 45.064 12.011 34.030 1.00 28.34 C ATOM1076 CD LYS 136 44.423 12.287 32.670 1.00 33.82 C ATOM 1077 CE LYS 13644.772 13.685 32.158 1.00 35.73 C ATOM 1078 NZ LYS 136 43.909 14.10831.012 1.00 37.92 N ATOM 1079 C LYS 136 42.558 9.214 35.118 1.00 21.60 CATOM 1080 O LYS 136 42.430 8.227 34.398 1.00 20.22 O ATOM 1081 N LYS 13742.306 9.189 36.423 1.00 18.95 N ATOM 1082 CA LYS 137 41.852 7.97437.090 1.00 21.28 C ATOM 1083 CB LYS 137 41.544 8.273 38.562 1.00 25.63C ATOM 1084 CG LYS 137 41.269 7.050 39.426 1.00 31.71 C ATOM 1085 CD LYS137 42.537 6.254 39.686 1.00 35.51 C ATOM 1086 CE LYS 137 42.304 5.15240.715 1.00 38.22 C ATOM 1087 NZ LYS 137 41.271 4.180 40.265 1.00 40.14N ATOM 1088 C LYS 137 40.598 7.438 36.392 1.00 20.35 C ATOM 1089 O LYS137 40.521 6.256 36.058 1.00 20.90 O ATOM 1090 N TYR 138 39.615 8.30836.170 1.00 20.99 N ATOM 1091 CA TYR 138 38.389 7.891 35.504 1.00 19.67C ATOM 1092 CB TYR 138 37.304 8.954 35.669 1.00 23.50 C ATOM 1093 CG TYR138 36.562 8.834 36.982 1.00 26.68 C ATOM 1094 CD1 TYR 138 36.955 9.56238.109 1.00 28.98 C ATOM 1095 CE1 TYR 138 36.274 9.429 39.325 1.00 30.78C ATOM 1096 CD2 TYR 138 35.478 7.970 37.103 1.00 27.96 C ATOM 1097 CE2TYR 138 34.797 7.826 38.307 1.00 29.85 C ATOM 1098 CZ TYR 138 35.1958.553 39.411 1.00 32.30 C ATOM 1099 OH TYR 138 34.513 8.389 40.594 1.0032.94 O ATOM 1100 C TYR 138 38.602 7.563 34.027 1.00 19.85 C ATOM 1101 OTYR 138 37.943 6.676 33.477 1.00 18.93 O ATOM 1102 N LEU 139 39.5228.277 33.386 1.00 19.83 N ATOM 1103 CA LEU 139 39.828 8.022 31.983 1.0019.04 C ATOM 1104 CB LEU 139 40.925 8.977 31.504 1.00 17.82 C ATOM 1105CG LEU 139 41.430 8.814 30.065 1.00 19.51 C ATOM 1106 CD1 LEU 139 40.2528.806 29.088 1.00 20.02 C ATOM 1107 CD2 LEU 139 42.399 9.941 29.740 1.0019.89 C ATOM 1108 C LEU 139 40.309 6.574 31.898 1.00 18.34 C ATOM 1109 OLEU 139 39.940 5.827 30.991 1.00 18.31 O ATOM 1110 N TYR 140 41.1256.185 32.872 1.00 17.67 N ATOM 1111 CA TYR 140 41.660 4.830 32.950 1.0017.75 C ATOM 1112 CB TYR 140 42.751 4.787 34.039 1.00 17.17 C ATOM 1113CG TYR 140 42.900 3.472 34.770 1.00 18.96 C ATOM 1114 CD1 TYR 140 42.1793.220 35.933 1.00 16.92 C ATOM 1115 CE1 TYR 140 42.309 2.017 36.614 1.0019.26 C ATOM 1116 CD2 TYR 140 43.764 2.482 34.301 1.00 17.81 C ATOM 1117CE2 TYR 140 43.902 1.276 34.976 1.00 19.95 C ATOM 1118 CZ TYR 140 43.1701.051 36.131 1.00 18.94 C ATOM 1119 OH TYR 140 43.282 −0.143 36.793 1.0019.16 O ATOM 1120 C TYR 140 40.554 3.802 33.225 1.00 17.59 C ATOM 1121 OTYR 140 40.525 2.734 32.615 1.00 17.88 O ATOM 1122 N GLU 141 39.6334.131 34.124 1.00 16.76 N ATOM 1123 CA GLU 141 38.544 3.213 34.464 1.0017.55 C ATOM 1124 CB GLU 141 37.711 3.770 35.625 1.00 18.26 C ATOM 1125CG GLU 141 38.487 4.002 36.914 1.00 20.55 C ATOM 1126 CD GLU 141 38.8362.718 37.653 1.00 20.42 C ATOM 1127 OE1 GLU 141 38.589 1.614 37.127 1.0022.10 O ATOM 1128 OE2 GLU 141 39.372 2.819 38.773 1.00 23.02 O ATOM 1129C GLU 141 37.630 2.955 33.269 1.00 17.54 C ATOM 1130 O GLU 141 37.1631.832 33.048 1.00 15.35 O ATOM 1131 N ILE 142 37.376 3.996 32.489 1.0017.68 N ATOM 1132 CA ILE 142 36.508 3.851 31.329 1.00 18.05 C ATOM 1133CB ILE 142 36.002 5.227 30.836 1.00 16.78 C ATOM 1134 CG2 ILE 142 35.3235.091 29.474 1.00 18.86 C ATOM 1135 CG1 ILE 142 34.983 5.780 31.832 1.0017.88 C ATOM 1136 CD1 ILE 142 33.767 4.879 32.000 1.00 19.63 C ATOM 1137C ILE 142 37.217 3.134 30.193 1.00 17.02 C ATOM 1138 O ILE 142 36.6392.262 29.547 1.00 17.44 O ATOM 1139 N ALA 143 38.466 3.513 29.946 1.0017.64 N ATOM 1140 CA ALA 143 39.251 2.907 28.877 1.00 19.82 C ATOM 1141CB ALA 143 40.641 3.552 28.811 1.00 18.81 C ATOM 1142 C ALA 143 39.3911.396 29.057 1.00 20.58 C ATOM 1143 O ALA 143 39.113 0.633 28.136 1.0022.08 O ATOM 1144 N ARG 144 39.814 0.959 30.239 1.00 20.57 N ATOM 1145CA ARG 144 39.990 −0.471 30.457 1.00 21.41 C ATOM 1146 CB ARG 144 40.738−0.726 31.763 1.00 20.26 C ATOM 1147 CG ARG 144 39.956 −0.441 33.0341.00 22.71 C ATOM 1148 CD ARG 144 40.912 −0.524 34.208 1.00 23.88 C ATOM1149 NE ARG 144 40.244 −0.438 35.498 1.00 27.58 N ATOM 1150 CZ ARG 14440.340 −1.371 36.441 1.00 29.71 C ATOM 1151 NH1 ARG 144 39.705 −1.21737.596 1.00 30.74 N ATOM 1152 NH2 ARG 144 41.062 −2.462 36.226 1.0029.23 N ATOM 1153 C ARG 144 38.679 −1.257 30.432 1.00 20.40 C ATOM 1154O ARG 144 38.686 −2.467 30.235 1.00 23.27 O ATOM 1155 N ARG 145 37.558−0.573 30.634 1.00 20.53 N ATOM 1156 CA ARG 145 36.250 −1.223 30.6041.00 21.62 C ATOM 1157 CB ARG 145 35.250 −0.499 31.507 1.00 21.06 C ATOM1158 CG ARG 145 35.400 −0.802 32.973 1.00 23.28 C ATOM 1159 CD ARG 14534.334 −0.078 33.780 1.00 23.01 C ATOM 1160 NE ARG 145 34.398 −0.44335.189 1.00 23.19 N ATOM 1161 CZ ARG 145 35.425 −0.165 35.982 1.00 26.04C ATOM 1162 NH1 ARG 145 35.399 −0.535 37.252 1.00 23.21 N ATOM 1163 NH2ARG 145 36.482 0.487 35.502 1.00 24.21 N ATOM 1164 C ARG 145 35.711−1.209 29.187 1.00 22.10 C ATOM 1165 O ARG 145 34.767 −1.931 28.862 1.0021.98 O ATOM 1166 N HIS 146 36.304 −0.364 28.352 1.00 20.74 N ATOM 1167CA HIS 146 35.883 −0.252 26.975 1.00 21.51 C ATOM 1168 CB HIS 146 35.0411.007 26.781 1.00 22.50 C ATOM 1169 CG HIS 146 33.792 1.010 27.598 1.0022.49 C ATOM 1170 CD2 HIS 146 32.540 0.588 27.309 1.00 22.56 C ATOM 1171ND1 HIS 146 33.771 1.401 28.919 1.00 24.88 N ATOM 1172 CE1 HIS 14632.560 1.216 29.410 1.00 22.26 C ATOM 1173 NE2 HIS 146 31.794 0.72228.453 1.00 26.94 N ATOM 1174 C HIS 146 37.037 −0.260 25.995 1.00 24.46C ATOM 1175 O HIS 146 37.323 0.744 25.338 1.00 25.17 O ATOM 1176 N PRO147 37.744 −1.392 25.904 1.00 26.06 N ATOM 1177 CD PRO 147 37.460 −2.73726.437 1.00 24.64 C ATOM 1178 CA PRO 147 38.851 −1.419 24.951 1.00 28.42C ATOM 1179 CB PRO 147 39.485 −2.776 25.218 1.00 27.61 C ATOM 1180 CGPRO 147 38.280 −3.633 25.526 1.00 25.89 C ATOM 1181 C PRO 147 38.085−1.374 23.636 1.00 30.98 C ATOM 1182 O PRO 147 37.006 −1.947 23.549 1.0036.58 O ATOM 1183 N TYR 148 38.615 −0.688 22.638 1.00 32.85 N ATOM 1184CA TYR 148 37.954 −0.545 21.334 1.00 29.99 C ATOM 1185 CB TYR 148 36.941−1.675 21.054 1.00 33.17 C ATOM 1186 CG TYR 148 37.557 −3.053 20.9391.00 35.18 C ATOM 1187 CD1 TYR 148 38.635 −3.291 20.085 1.00 36.97 CATOM 1188 CE1 TYR 148 39.216 −4.555 19.996 1.00 38.30 C ATOM 1189 CD2TYR 148 37.072 −4.116 21.698 1.00 36.29 C ATOM 1190 CE2 TYR 148 37.643−5.378 21.617 1.00 36.22 C ATOM 1191 CZ TYR 148 38.712 −5.592 20.7691.00 37.87 C ATOM 1192 OH TYR 148 39.282 −6.843 20.711 1.00 39.48 O ATOM1193 C TYR 148 37.251 0.803 21.237 1.00 27.72 C ATOM 1194 O TYR 14836.952 1.262 20.138 1.00 26.55 O ATOM 1195 N PHE 149 36.974 1.439 22.3751.00 24.34 N ATOM 1196 CA PHE 149 36.345 2.761 22.340 1.00 21.61 C ATOM1197 CB PHE 149 35.929 3.210 23.745 1.00 20.84 C ATOM 1198 CG PHE 14934.976 4.382 23.760 1.00 21.56 C ATOM 1199 CD1 PHE 149 35.361 5.62523.262 1.00 22.58 C ATOM 1200 CD2 PHE 149 33.689 4.238 24.272 1.00 24.69C ATOM 1201 CE1 PHE 149 34.482 6.705 23.274 1.00 23.37 C ATOM 1202 CE2PHE 149 32.796 5.314 24.288 1.00 21.99 C ATOM 1203 CZ PHE 149 33.1956.548 23.789 1.00 25.70 C ATOM 1204 C PHE 149 37.458 3.661 21.796 1.0020.57 C ATOM 1205 O PHE 149 38.537 3.744 22.382 1.00 18.38 O ATOM 1206 NTYR 150 37.202 4.307 20.664 1.00 18.85 N ATOM 1207 CA TYR 150 38.1915.173 20.023 1.00 19.13 C ATOM 1208 CB TYR 150 37.527 5.960 18.893 1.0018.20 C ATOM 1209 CG TYR 150 38.509 6.627 17.963 1.00 17.36 C ATOM 1210CD1 TYR 150 38.404 7.984 17.673 1.00 18.89 C ATOM 1211 CE1 TYR 15039.339 8.621 16.850 1.00 18.93 C ATOM 1212 CD2 TYR 150 39.568 5.90917.403 1.00 17.97 C ATOM 1213 CE2 TYR 150 40.510 6.537 16.578 1.00 19.15C ATOM 1214 CZ TYR 150 40.387 7.892 16.312 1.00 19.04 C ATOM 1215 OH TYR150 41.333 8.531 15.540 1.00 19.32 O ATOM 1216 C TYR 150 38.832 6.12621.038 1.00 19.02 C ATOM 1217 O TYR 150 38.177 7.038 21.549 1.00 17.89 OATOM 1218 N ALA 151 40.119 5.915 21.307 1.00 18.89 N ATOM 1219 CA ALA151 40.855 6.712 22.289 1.00 18.55 C ATOM 1220 CB ALA 151 42.343 6.35522.243 1.00 18.96 C ATOM 1221 C ALA 151 40.685 8.228 22.221 1.00 18.61 CATOM 1222 O ALA 151 40.335 8.858 23.218 1.00 17.01 O ATOM 1223 N PRO 15240.939 8.836 21.054 1.00 20.09 N ATOM 1224 CD PRO 152 41.443 8.26619.791 1.00 20.43 C ATOM 1225 CA PRO 152 40.794 10.289 20.941 1.00 18.74C ATOM 1226 CB PRO 152 41.043 10.542 19.457 1.00 20.71 C ATOM 1227 CGPRO 152 42.032 9.484 19.109 1.00 18.05 C ATOM 1228 C PRO 152 39.42310.767 21.386 1.00 19.54 C ATOM 1229 O PRO 152 39.290 11.819 22.016 1.0017.71 O ATOM 1230 N GLU 153 38.401 9.977 21.075 1.00 19.85 N ATOM 1231CA GLU 153 37.055 10.359 21.444 1.00 21.95 C ATOM 1232 CB GLU 153 36.0339.387 20.849 1.00 24.96 C ATOM 1233 CG GLU 153 34.650 9.992 20.756 1.0031.24 C ATOM 1234 CD GLU 153 33.597 8.991 20.348 1.00 35.06 C ATOM 1235OE1 GLU 153 33.846 8.211 19.403 1.00 38.93 O ATOM 1236 OE2 GLU 15332.515 8.994 20.969 1.00 36.93 O ATOM 1237 C GLU 153 36.922 10.41122.961 1.00 20.85 C ATOM 1238 O GLU 153 36.189 11.239 23.492 1.00 18.84O ATOM 1239 N LEU 154 37.629 9.534 23.667 1.00 20.47 N ATOM 1240 CA LEU154 37.568 9.559 25.126 1.00 20.34 C ATOM 1241 CB LEU 154 38.336 8.38325.720 1.00 20.20 C ATOM 1242 CG LEU 154 37.714 7.010 25.482 1.00 18.80C ATOM 1243 CD1 LEU 154 38.639 5.932 26.025 1.00 20.39 C ATOM 1244 CD2LEU 154 36.357 6.939 26.166 1.00 19.78 C ATOM 1245 C LEU 154 38.16010.866 25.637 1.00 20.24 C ATOM 1246 O LEU 154 37.769 11.366 26.684 1.0019.41 O ATOM 1247 N LEU 155 39.103 11.420 24.884 1.00 21.90 N ATOM 1248CA LEU 155 39.740 12.677 25.261 1.00 24.33 C ATOM 1249 CB LEU 155 40.93612.946 24.348 1.00 27.49 C ATOM 1250 CG LEU 155 42.301 12.999 25.0351.00 30.37 C ATOM 1251 CD1 LEU 155 42.503 11.765 25.902 1.00 29.59 CATOM 1252 CD2 LEU 155 43.387 13.106 23.975 1.00 29.50 C ATOM 1253 C LEU155 38.743 13.829 25.168 1.00 24.16 C ATOM 1254 O LEU 155 38.797 14.78525.953 1.00 22.95 O ATOM 1255 N PHE 156 37.836 13.743 24.201 1.00 22.87N ATOM 1256 CA PHE 156 36.826 14.778 24.036 1.00 23.49 C ATOM 1257 CBPHE 156 36.090 14.589 22.708 1.00 25.45 C ATOM 1258 CG PHE 156 36.93214.928 21.503 1.00 29.56 C ATOM 1259 CD1 PHE 156 37.151 16.255 21.1441.00 31.82 C ATOM 1260 CD2 PHE 156 37.533 13.922 20.749 1.00 30.99 CATOM 1261 CE1 PHE 156 37.960 16.577 20.050 1.00 33.40 C ATOM 1262 CE2PHE 156 38.344 14.229 19.654 1.00 30.51 C ATOM 1263 CZ PHE 156 38.55715.556 19.305 1.00 34.47 C ATOM 1264 C PHE 156 35.859 14.714 25.213 1.0022.22 C ATOM 1265 O PHE 156 35.474 15.738 25.759 1.00 22.45 O ATOM 1266N PHE 157 35.466 13.509 25.608 1.00 22.20 N ATOM 1267 CA PHE 157 34.57013.365 26.756 1.00 22.08 C ATOM 1268 CB PHE 157 34.214 11.894 26.9671.00 22.26 C ATOM 1269 CG PHE 157 33.025 11.442 26.182 1.00 23.20 C ATOM1270 CD1 PHE 157 31.738 11.705 26.637 1.00 24.43 C ATOM 1271 CD2 PHE 15733.186 10.772 24.974 1.00 22.58 C ATOM 1272 CE1 PHE 157 30.623 11.30825.896 1.00 22.53 C ATOM 1273 CE2 PHE 157 32.083 10.373 24.229 1.0023.96 C ATOM 1274 CZ PHE 157 30.799 10.642 24.690 1.00 24.03 C ATOM 1275C PHE 157 35.245 13.908 28.019 1.00 22.30 C ATOM 1276 O PHE 157 34.62914.625 28.810 1.00 21.71 O ATOM 1277 N ALA 158 36.521 13.564 28.191 1.0023.15 N ATOM 1278 CA ALA 158 37.296 13.990 29.353 1.00 23.32 C ATOM 1279CB ALA 158 38.724 13.486 29.233 1.00 26.20 C ATOM 1280 C ALA 158 37.29515.497 29.531 1.00 22.66 C ATOM 1281 O ALA 158 37.059 16.002 30.629 1.0020.82 O ATOM 1282 N LYS 159 37.567 16.212 28.446 1.00 22.65 N ATOM 1283CA LYS 159 37.599 17.663 28.482 1.00 23.96 C ATOM 1284 CB LYS 159 38.10418.194 27.136 1.00 28.43 C ATOM 1285 CG LYS 159 39.526 17.712 26.8451.00 33.70 C ATOM 1286 CD LYS 159 40.002 17.957 25.421 1.00 36.49 C ATOM1287 CE LYS 159 41.387 17.324 25.234 1.00 36.49 C ATOM 1288 NZ LYS 15941.914 17.388 23.834 1.00 39.43 N ATOM 1289 C LYS 159 36.220 18.20928.822 1.00 23.61 C ATOM 1290 O LYS 159 36.096 19.269 29.426 1.00 22.68O ATOM 1291 N ARG 160 35.179 17.478 28.441 1.00 22.77 N ATOM 1292 CA ARG160 33.828 17.914 28.751 1.00 23.39 C ATOM 1293 CB ARG 160 32.819 17.20127.844 1.00 24.29 C ATOM 1294 CG ARG 160 32.937 17.636 26.383 1.00 28.21C ATOM 1295 CD ARG 160 31.916 16.937 25.508 1.00 30.52 C ATOM 1296 NEARG 160 30.559 17.165 25.986 1.00 32.81 N ATOM 1297 CZ ARG 160 29.49916.487 25.563 1.00 35.18 C ATOM 1298 NH1 ARG 160 28.298 16.761 26.0541.00 34.50 N ATOM 1299 NH2 ARG 160 29.643 15.529 24.655 1.00 33.24 NATOM 1300 C ARG 160 33.526 17.657 30.227 1.00 22.94 C ATOM 1301 O ARG160 32.819 18.438 30.859 1.00 23.10 O ATOM 1302 N TYR 161 34.055 16.57030.787 1.00 22.17 N ATOM 1303 CA TYR 161 33.826 16.304 32.207 1.00 22.32C ATOM 1304 CB TYR 161 34.322 14.921 32.617 1.00 22.26 C ATOM 1305 CGTYR 161 33.319 13.812 32.437 1.00 22.40 C ATOM 1306 CD1 TYR 161 33.20813.137 31.224 1.00 23.51 C ATOM 1307 CE1 TYR 161 32.324 12.062 31.0771.00 23.70 C ATOM 1308 CD2 TYR 161 32.512 13.396 33.500 1.00 21.24 CATOM 1309 CE2 TYR 161 31.629 12.327 33.358 1.00 23.56 C ATOM 1310 CZ TYR161 31.546 11.661 32.146 1.00 23.86 C ATOM 1311 OH TYR 161 30.725 10.55832.016 1.00 24.68 O ATOM 1312 C TYR 161 34.584 17.337 33.017 1.00 22.45C ATOM 1313 O TYR 161 34.088 17.848 34.021 1.00 23.97 O ATOM 1314 N LYS162 35.800 17.633 32.581 1.00 22.47 N ATOM 1315 CA LYS 162 36.622 18.60933.269 1.00 23.35 C ATOM 1316 CB LYS 162 37.949 18.796 32.537 1.00 25.48C ATOM 1317 CG LYS 162 38.951 19.677 33.275 1.00 29.28 C ATOM 1318 CDLYS 162 40.323 19.592 32.619 1.00 32.83 C ATOM 1319 CE LYS 162 41.37320.335 33.424 1.00 34.63 C ATOM 1320 NZ LYS 162 42.733 20.156 32.8441.00 37.31 N ATOM 1321 C LYS 162 35.867 19.929 33.351 1.00 24.15 C ATOM1322 O LYS 162 35.819 20.551 34.407 1.00 23.68 O ATOM 1323 N ALA 16335.255 20.340 32.243 1.00 23.61 N ATOM 1324 CA ALA 163 34.496 21.58632.217 1.00 24.66 C ATOM 1325 CB ALA 163 34.029 21.888 30.793 1.00 25.16C ATOM 1326 C ALA 163 33.293 21.531 33.155 1.00 25.18 C ATOM 1327 O ALA163 32.994 22.506 33.851 1.00 23.19 O ATOM 1328 N ALA 164 32.602 20.39233.161 1.00 24.67 N ATOM 1329 CA ALA 164 31.434 20.206 34.011 1.00 23.89C ATOM 1330 CB ALA 164 30.837 18.823 33.784 1.00 24.91 C ATOM 1331 C ALA164 31.805 20.378 35.480 1.00 24.76 C ATOM 1332 O ALA 164 31.129 21.08436.217 1.00 24.38 O ATOM 1333 N PHE 165 32.879 19.723 35.901 1.00 23.75N ATOM 1334 CA PHE 165 33.330 19.816 37.284 1.00 25.72 C ATOM 1335 CBPHE 165 34.400 18.760 37.550 1.00 24.54 C ATOM 1336 CG PHE 165 33.83717.403 37.824 1.00 26.46 C ATOM 1337 CD1 PHE 165 33.280 17.110 39.0641.00 27.51 C ATOM 1338 CD2 PHE 165 33.829 16.426 36.839 1.00 24.70 CATOM 1339 CE1 PHE 165 32.722 15.859 39.318 1.00 27.32 C ATOM 1340 CE2PHE 165 33.272 15.172 37.084 1.00 25.28 C ATOM 1341 CZ PHE 165 32.71914.890 38.325 1.00 26.20 C ATOM 1342 C PHE 165 33.868 21.194 37.639 1.0027.02 C ATOM 1343 O PHE 165 33.647 21.690 38.747 1.00 26.95 O ATOM 1344N THR 166 34.576 21.806 36.696 1.00 26.67 N ATOM 1345 CA THR 166 35.14623.128 36.910 1.00 28.42 C ATOM 1346 CB THR 166 35.966 23.573 35.6881.00 28.67 C ATOM 1347 OG1 THR 166 37.099 22.710 35.541 1.00 28.72 OATOM 1348 CG2 THR 166 36.438 25.017 35.856 1.00 30.22 C ATOM 1349 C THR166 34.057 24.159 37.173 1.00 27.78 C ATOM 1350 O THR 166 34.162 24.97038.094 1.00 29.29 O ATOM 1351 N GLU 167 33.008 24.114 36.365 1.00 28.67N ATOM 1352 CA GLU 167 31.905 25.048 36.498 1.00 29.33 C ATOM 1353 CBGLU 167 31.068 25.070 35.219 1.00 30.56 C ATOM 1354 CG GLU 167 29.76625.841 35.382 1.00 32.73 C ATOM 1355 CD GLU 167 28.817 25.673 34.2181.00 33.21 C ATOM 1356 OE1 GLU 167 27.734 26.295 34.245 1.00 36.42 OATOM 1357 OE2 GLU 167 29.146 24.924 33.278 1.00 32.03 O ATOM 1358 C GLU167 30.975 24.731 37.657 1.00 30.62 C ATOM 1359 O GLU 167 30.626 25.60238.458 1.00 28.60 O ATOM 1360 N CYS 168 30.568 23.473 37.735 1.00 27.70N ATOM 1361 CA CYS 168 29.630 23.054 38.752 1.00 29.04 C ATOM 1362 C CYS168 30.121 22.921 40.179 1.00 28.27 C ATOM 1363 O CYS 168 29.335 23.06141.114 1.00 29.41 O ATOM 1364 CB CYS 168 28.977 21.752 38.318 1.00 28.12C ATOM 1365 SG CYS 168 27.921 21.910 36.848 1.00 27.31 S ATOM 1366 N CYS169 31.405 22.662 40.374 1.00 27.98 N ATOM 1367 CA CYS 169 31.888 22.50741.734 1.00 29.91 C ATOM 1368 C CYS 169 32.060 23.801 42.516 1.00 32.10C ATOM 1369 O CYS 169 32.329 23.769 43.713 1.00 32.90 O ATOM 1370 CB CYS169 33.185 21.717 41.742 1.00 28.75 C ATOM 1371 SG CYS 169 32.940 19.96041.320 1.00 27.95 S ATOM 1372 N GLN 170 31.914 24.937 41.844 1.00 34.07N ATOM 1373 CA GLN 170 32.039 26.225 42.516 1.00 36.89 C ATOM 1374 CBGLN 170 33.235 27.013 41.967 1.00 39.05 C ATOM 1375 CG GLN 170 33.35927.028 40.453 1.00 43.03 C ATOM 1376 CD GLN 170 34.626 27.730 39.9871.00 44.37 C ATOM 1377 OE1 GLN 170 34.748 28.952 40.094 1.00 45.73 OATOM 1378 NE2 GLN 170 35.580 26.957 39.478 1.00 44.70 N ATOM 1379 C GLN170 30.753 27.018 42.348 1.00 36.48 C ATOM 1380 O GLN 170 30.720 28.23242.546 1.00 36.94 O ATOM 1381 N ALA 171 29.689 26.313 41.986 1.00 35.53N ATOM 1382 CA ALA 171 28.391 26.940 41.799 1.00 36.43 C ATOM 1383 CBALA 171 27.594 26.183 40.746 1.00 34.62 C ATOM 1384 C ALA 171 27.63626.947 43.125 1.00 36.32 C ATOM 1385 O ALA 171 28.048 26.294 44.085 1.0035.91 O ATOM 1386 N ALA 172 26.536 27.691 43.171 1.00 36.11 N ATOM 1387CA ALA 172 25.718 27.769 44.374 1.00 37.38 C ATOM 1388 CB ALA 172 24.66728.860 44.225 1.00 37.30 C ATOM 1389 C ALA 172 25.047 26.423 44.604 1.0036.99 C ATOM 1390 O ALA 172 25.080 25.880 45.709 1.00 37.10 O ATOM 1391N ASP 173 24.435 25.885 43.555 1.00 36.21 N ATOM 1392 CA ASP 173 23.77424.594 43.657 1.00 35.77 C ATOM 1393 CB ASP 173 22.340 24.683 43.1441.00 36.43 C ATOM 1394 CG ASP 173 21.530 23.448 43.476 1.00 39.19 C ATOM1395 OD1 ASP 173 20.301 23.463 43.251 1.00 41.76 O ATOM 1396 OD2 ASP 17322.123 22.456 43.958 1.00 39.06 O ATOM 1397 C ASP 173 24.564 23.58242.840 1.00 34.19 C ATOM 1398 O ASP 173 24.194 23.244 41.722 1.00 34.19O ATOM 1399 N LYS 174 25.660 23.111 43.422 1.00 33.55 N ATOM 1400 CA LYS174 26.547 22.153 42.778 1.00 32.09 C ATOM 1401 CB LYS 174 27.558 21.62243.800 1.00 31.57 C ATOM 1402 CG LYS 174 28.439 22.719 44.383 1.00 33.11C ATOM 1403 CD LYS 174 29.484 22.192 45.355 1.00 34.10 C ATOM 1404 CELYS 174 30.330 23.339 45.913 1.00 34.70 C ATOM 1405 NZ LYS 174 31.43822.862 46.785 1.00 32.16 N ATOM 1406 C LYS 174 25.833 20.997 42.084 1.0031.19 C ATOM 1407 O LYS 174 26.006 20.797 40.884 1.00 29.30 O ATOM 1408N ALA 175 25.019 20.253 42.827 1.00 29.90 N ATOM 1409 CA ALA 175 24.31019.115 42.257 1.00 30.27 C ATOM 1410 CB ALA 175 23.528 18.379 43.3481.00 31.51 C ATOM 1411 C ALA 175 23.379 19.472 41.104 1.00 31.06 C ATOM1412 O ALA 175 23.363 18.787 40.079 1.00 30.11 O ATOM 1413 N ALA 17622.599 20.537 41.268 1.00 30.08 N ATOM 1414 CA ALA 176 21.661 20.95440.229 1.00 31.61 C ATOM 1415 CB ALA 176 20.908 22.200 40.678 1.00 32.83C ATOM 1416 C ALA 176 22.375 21.229 38.911 1.00 30.95 C ATOM 1417 O ALA176 21.825 21.022 37.832 1.00 31.05 O ATOM 1418 N CYS 177 23.607 21.69739.022 1.00 31.02 N ATOM 1419 CA CYS 177 24.441 22.027 37.875 1.00 31.16C ATOM 1420 C CYS 177 25.154 20.794 37.330 1.00 29.17 C ATOM 1421 O CYS177 25.102 20.504 36.135 1.00 26.73 O ATOM 1422 CB CYS 177 25.473 23.06338.327 1.00 31.83 C ATOM 1423 SG CYS 177 26.816 23.583 37.205 1.00 34.69S ATOM 1424 N LEU 178 25.811 20.075 38.234 1.00 27.72 N ATOM 1425 CA LEU178 26.602 18.896 37.896 1.00 26.57 C ATOM 1426 CB LEU 178 27.447 18.48939.108 1.00 25.91 C ATOM 1427 CG LEU 178 28.925 18.144 38.896 1.00 28.89C ATOM 1428 CD1 LEU 178 29.349 17.175 39.988 1.00 25.58 C ATOM 1429 CD2LEU 178 29.167 17.538 37.523 1.00 26.61 C ATOM 1430 C LEU 178 25.88117.651 37.380 1.00 24.80 C ATOM 1431 O LEU 178 26.206 17.149 36.303 1.0025.41 O ATOM 1432 N LEU 179 24.924 17.144 38.149 1.00 22.68 N ATOM 1433CA LEU 179 24.217 15.922 37.775 1.00 24.35 C ATOM 1434 CB LEU 179 23.12515.594 38.803 1.00 24.85 C ATOM 1435 CG LEU 179 23.638 15.145 40.1761.00 25.93 C ATOM 1436 CD1 LEU 179 24.669 14.039 40.026 1.00 27.87 CATOM 1437 CD2 LEU 179 24.264 16.300 40.878 1.00 31.20 C ATOM 1438 C LEU179 23.644 15.872 36.366 1.00 24.16 C ATOM 1439 O LEU 179 23.842 14.89135.656 1.00 23.34 O ATOM 1440 N PRO 180 22.918 16.915 35.940 1.00 24.79N ATOM 1441 CD PRO 180 22.363 18.077 36.660 1.00 26.35 C ATOM 1442 CAPRO 180 22.387 16.841 34.578 1.00 25.66 C ATOM 1443 CB PRO 180 21.63718.163 34.431 1.00 27.05 C ATOM 1444 CG PRO 180 21.135 18.410 35.8261.00 27.13 C ATOM 1445 C PRO 180 23.514 16.692 33.554 1.00 24.94 C ATOM1446 O PRO 180 23.363 16.016 32.543 1.00 23.29 O ATOM 1447 N LYS 18124.648 17.330 33.821 1.00 25.66 N ATOM 1448 CA LYS 181 25.775 17.24832.904 1.00 26.06 C ATOM 1449 CB LYS 181 26.833 18.294 33.268 1.00 26.88C ATOM 1450 CG LYS 181 26.350 19.730 33.099 1.00 29.31 C ATOM 1451 CDLYS 181 27.406 20.737 33.521 1.00 30.97 C ATOM 1452 CE LYS 181 26.90022.166 33.354 1.00 31.56 C ATOM 1453 NZ LYS 181 26.605 22.490 31.9301.00 34.87 N ATOM 1454 C LYS 181 26.378 15.845 32.919 1.00 24.07 C ATOM1455 O LYS 181 26.760 15.318 31.877 1.00 23.35 O ATOM 1456 N LEU 18226.459 15.243 34.101 1.00 24.47 N ATOM 1457 CA LEU 182 27.006 13.89634.225 1.00 25.75 C ATOM 1458 CB LEU 182 27.220 13.532 35.698 1.00 24.72C ATOM 1459 CG LEU 182 28.266 14.366 36.448 1.00 24.56 C ATOM 1460 CD1LEU 182 28.369 13.893 37.886 1.00 24.18 C ATOM 1461 CD2 LEU 182 29.61314.242 35.755 1.00 23.06 C ATOM 1462 C LEU 182 26.053 12.903 33.565 1.0026.04 C ATOM 1463 O LEU 182 26.487 11.927 32.949 1.00 25.29 O ATOM 1464N ASP 183 24.755 13.158 33.698 1.56 27.42 N ATOM 1465 CA ASP 183 23.75112.302 33.082 1.00 28.92 C ATOM 1466 CB ASP 183 22.337 12.784 33.4241.00 30.09 C ATOM 1467 CG ASP 183 21.888 12.378 34.819 1.00 32.33 C ATOM1468 OD1 ASP 183 20.739 12.716 35.181 1.00 33.14 O ATOM 1469 OD2 ASP 18322.666 11.727 35.552 1.00 32.75 O ATOM 1470 C ASP 183 23.941 12.35031.566 1.00 28.35 C ATOM 1471 O ASP 183 23.942 11.313 30.904 1.00 29.41O ATOM 1472 N GLU 184 24.095 13.557 31.023 1.00 28.13 N ATOM 1473 CA GLU184 24.287 13.730 29.584 1.00 29.50 C ATOM 1474 CB GLU 184 24.382 15.21329.216 1.00 32.17 C ATOM 1475 CG GLU 184 23.108 16.013 29.433 1.00 38.93C ATOM 1476 CD GLU 184 23.186 17.414 28.830 1.00 43.43 C ATOM 1477 OE1GLU 184 22.236 18.208 29.029 1.00 45.37 O ATOM 1478 OE2 GLU 184 24.19517.721 28.153 1.00 44.53 O ATOM 1479 C GLU 184 25.546 13.013 29.095 1.0027.76 C ATOM 1480 O GLU 184 25.486 12.215 28.162 1.00 28.37 O ATOM 1481N LEU 185 26.682 13.299 29.726 1.00 26.01 N ATOM 1482 CA LEU 185 27.94612.665 29.351 1.00 24.13 C ATOM 1483 CB LEU 185 29.068 13.097 30.3011.00 23.78 C ATOM 1484 CG LEU 185 29.920 14.337 30.011 1.00 26.40 C ATOM1485 CD1 LEU 185 29.773 14.749 28.557 1.00 23.77 C ATOM 1486 CD2 LEU 18529.531 15.458 30.947 1.00 24.62 C ATOM 1487 C LEU 185 27.835 11.14529.388 1.00 23.55 C ATOM 1488 O LEU 185 28.290 10.447 28.482 1.00 24.27O ATOM 1489 N ARG 186 27.246 10.637 30.461 1.00 23.87 N ATOM 1490 CA ARG186 27.062 9.205 30.634 1.00 25.88 C ATOM 1491 CB ARG 186 26.352 8.95931.969 1.00 29.61 C ATOM 1492 CG ARG 186 26.140 7.514 32.363 1.00 33.22C ATOM 1493 CD ARG 186 24.849 6.945 31.803 1.00 37.79 C ATOM 1494 NE ARG186 23.697 7.810 32.055 1.00 41.24 N ATOM 1495 CZ ARG 186 23.229 8.13433.258 1.00 40.76 C ATOM 1496 NH1 ARG 186 22.174 8.929 33.361 1.00 41.85N ATOM 1497 NH2 ARG 186 23.805 7.669 34.356 1.00 42.94 N ATOM 1498 C ARG186 26.254 8.628 29.471 1.00 26.22 C ATOM 1499 O ARG 186 26.642 7.61728.868 1.00 24.27 O ATOM 1500 N ASP 187 25.137 9.276 29.153 1.00 25.62 NATOM 1501 CA ASP 187 24.283 8.813 28.067 1.00 28.35 C ATOM 1502 CB ASP187 22.992 9.645 27.985 1.00 29.91 C ATOM 1503 CG ASP 187 22.127 9.51729.233 1.00 33.76 C ATOM 1504 OD1 ASP 187 22.072 8.414 29.818 1.00 33.68O ATOM 1505 OD2 ASP 187 21.489 10.521 29.622 1.00 36.36 O ATOM 1506 CASP 187 25.013 8.872 26.731 1.00 26.67 C ATOM 1507 O ASP 187 24.9907.912 25.968 1.00 25.93 O ATOM 1508 N GLU 188 25.652 10.000 26.443 1.0027.13 N ATOM 1509 CA GLU 188 26.379 10.138 25.185 1.00 28.40 C ATOM 1510CB GLU 188 27.022 11.520 25.075 1.00 30.92 C ATOM 1511 CG GLU 188 26.04712.648 24.849 1.00 34.96 C ATOM 1512 CD GLU 188 26.747 13.977 24.6551.00 36.94 C ATOM 1513 OE1 GLU 188 27.564 14.091 23.714 1.00 39.19 OATOM 1514 OE2 GLU 188 26.479 14.906 25.443 1.00 39.17 O ATOM 1515 C GLU188 27.461 9.073 25.090 1.00 26.53 C ATOM 1516 O GLU 188 27.699 8.50624.023 1.00 25.50 O ATOM 1517 N GLY 189 28.122 8.819 26.213 1.00 26.17 NATOM 1518 CA GLY 189 29.166 7.815 26.242 1.00 25.87 C ATOM 1519 C GLY189 28.638 6.440 25.881 1.00 26.92 C ATOM 1520 O GLY 189 29.176 5.76725.000 1.00 26.55 O ATOM 1521 N LYS 190 27.584 6.010 26.564 1.00 26.90 NATOM 1522 CA LYS 190 27.007 4.698 26.289 1.00 26.46 C ATOM 1523 CB LYS190 25.833 4.430 27.232 1.00 27.78 C ATOM 1524 CG LYS 190 26.173 4.66728.695 1.00 31.57 C ATOM 1525 CD LYS 190 25.014 4.309 29.605 1.00 34.60C ATOM 1526 CE LYS 190 25.091 2.853 30.022 1.00 36.99 C ATOM 1527 NZ LYS190 26.221 2.633 30.971 1.00 37.56 N ATOM 1528 C LYS 190 26.547 4.63224.839 1.00 25.07 C ATOM 1529 O LYS 190 26.793 3.645 24.148 1.00 25.14 OATOM 1530 N ALA 191 25.893 5.694 24.379 1.00 23.99 N ATOM 1531 CA ALA191 25.409 5.751 23.005 1.00 24.91 C ATOM 1532 CB ALA 191 24.663 7.06322.760 1.00 24.71 C ATOM 1533 C ALA 191 26.564 5.617 22.019 1.00 24.18 CATOM 1534 O ALA 191 26.514 4.798 21.104 1.00 25.26 O ATOM 1535 N SER 19227.602 6.428 22.198 1.00 24.28 N ATOM 1536 CA SER 192 28.755 6.37221.309 1.00 22.65 C ATOM 1537 CB SER 192 29.838 7.345 21.777 1.00 24.88C ATOM 1538 OG SER 192 31.042 7.122 21.072 1.00 27.17 O ATOM 1539 C SER192 29.321 4.957 21.253 1.00 23.24 C ATOM 1540 O SER 192 29.632 4.44620.179 1.00 21.91 O ATOM 1541 N SER 193 29.446 4.319 22.411 1.00 24.41 NATOM 1542 CA SER 193 29.975 2.961 22.459 1.00 26.29 C ATOM 1543 CB SER193 30.124 2.509 23.911 1.00 26.81 C ATOM 1544 OG SER 193 30.633 1.18923.975 1.00 32.97 O ATOM 1545 C SER 193 29.050 2.000 21.702 1.00 27.89 CATOM 1546 O SER 193 29.511 1.156 20.926 1.00 27.93 O ATOM 1547 N ALA 19427.747 2.137 21.930 1.00 26.46 N ATOM 1548 CA ALA 194 26.754 1.29121.268 1.00 28.27 C ATOM 1549 CB ALA 194 25.355 1.606 21.808 1.00 25.08C ATOM 1550 C ALA 194 26.796 1.536 19.759 1.00 27.33 C ATOM 1551 O ALA194 26.775 0.599 18.963 1.00 28.34 O ATOM 1552 N LYS 195 26.849 2.80819.387 1.00 27.59 N ATOM 1553 CA LYS 195 26.894 3.218 17.991 1.00 29.17C ATOM 1554 CB LYS 195 26.910 4.741 17.909 1.00 30.90 C ATOM 1555 CG LYS195 26.945 5.293 16.500 1.00 33.88 C ATOM 1556 CD LYS 195 26.958 6.81616.515 1.00 38.36 C ATOM 1557 CE LYS 195 25.715 7.375 17.199 1.00 39.12C ATOM 1558 NZ LYS 195 25.656 8.863 17.127 1.00 41.92 N ATOM 1559 C LYS195 28.124 2.653 17.296 1.00 29.62 C ATOM 1560 O LYS 195 28.045 2.16516.162 1.00 28.45 O ATOM 1561 N GLN 196 29.263 2.725 17.977 1.00 28.61 NATOM 1562 CA GLN 196 30.511 2.209 17.423 1.00 29.56 C ATOM 1563 CB GLN196 31.680 2.458 18.398 1.00 28.99 C ATOM 1564 CG GLN 196 32.103 3.93018.525 1.00 31.76 C ATOM 1565 CD GLN 196 33.213 4.169 19.560 1.00 33.58C ATOM 1566 OE1 GLN 196 34.333 3.667 19.427 1.00 31.96 O ATOM 1567 NE2GLN 196 32.898 4.944 20.592 1.00 31.16 N ATOM 1568 C GLN 196 30.3680.714 17.137 1.00 29.06 C ATOM 1569 O GLN 196 30.825 0.227 16.107 1.0027.73 O ATOM 1570 N ARG 197 29.714 −0.011 18.039 1.00 30.08 N ATOM 1571CA ARG 197 29.542 −1.451 17.850 1.00 32.15 C ATOM 1572 CB ARG 197 29.176−2.128 19.175 1.00 34.50 C ATOM 1573 CG ARG 197 29.819 −3.503 19.3401.00 40.58 C ATOM 1574 CD ARG 197 28.795 −4.610 19.549 1.00 43.25 C ATOM1575 NE ARG 197 28.059 −4.461 20.804 1.00 46.41 N ATOM 1576 CZ ARG 19727.225 −5.373 21.297 1.00 47.58 C ATOM 1577 NH1 ARG 197 26.600 −5.15122.446 1.00 47.39 N ATOM 1578 NH2 ARG 197 27.019 −6.513 20.645 1.0048.93 N ATOM 1579 C ARG 197 28.486 −1.781 16.794 1.00 31.32 C ATOM 1580O ARG 197 28.658 −2.714 16.007 1.00 32.58 O ATOM 5145 FE HEM 605 32.3478.521 32.831 1.00 25.18 FE ATOM 5146 CHA HEM 605 29.989 6.250 31.8881.00 26.35 C ATOM 5147 CHB HEM 605 33.685 8.455 29.666 1.00 23.94 C ATOM5148 CHC HEM 605 34.763 10.734 33.777 1.00 25.27 C ATOM 5149 CHD HEM 60531.090 8.531 35.950 1.00 25.17 C ATOM 5150 NA HEM 605 31.911 7.56631.106 1.00 24.86 N ATOM 5151 C1A HEM 605 30.872 6.651 30.902 1.00 25.80C ATOM 5152 C2A HEM 605 30.847 6.163 29.510 1.00 27.42 C ATOM 5153 C3AHEM 605 31.993 6.866 28.870 1.00 25.02 C ATOM 5154 C4A HEM 605 32.5887.675 29.870 1.00 24.52 C ATOM 5155 CMA HEM 605 32.465 6.750 27.426 1.0024.96 C ATOM 5156 CAA HEM 605 29.882 5.186 28.930 1.00 29.17 C ATOM 5157CBA HEM 605 30.527 3.832 28.695 1.00 31.67 C ATOM 5158 CGA HEM 60529.518 2.774 28.376 1.00 33.06 C ATOM 5159 O1A HEM 605 29.021 2.07529.271 1.00 33.53 O ATOM 5160 O2A HEM 605 29.206 2.620 27.150 1.00 37.12O ATOM 5161 NB HEM 605 33.905 9.412 31.936 1.00 24.76 N ATOM 5162 C1BHEM 605 34.306 9.254 30.609 1.00 24.01 C ATOM 5163 C2B HEM 605 35.50910.069 30.357 1.00 25.66 C ATOM 5164 C3B HEM 605 35.803 10.700 31.4961.00 26.84 C ATOM 5165 C4B HEM 605 34.802 10.285 32.456 1.00 24.41 CATOM 5166 CMB HEM 605 36.214 10.115 29.023 1.00 25.60 C ATOM 5167 CABHEM 605 36.940 11.662 31.787 1.00 28.37 C ATOM 5168 CBB HEM 605 38.09511.278 32.280 1.00 31.00 C ATOM 5169 NC HEM 605 32.819 9.474 34.544 1.0024.53 N ATOM 5170 C1C HEM 605 33.853 10.372 34.756 1.00 25.60 C ATOM5171 C2C HEM 605 33.849 10.865 36.114 1.00 26.07 C ATOM 5172 C3C HEM 60532.809 10.235 36.722 1.00 26.20 C ATOM 5173 C4C HEM 605 32.185 9.36735.727 1.00 25.78 C ATOM 5174 CMC HEM 605 34.846 11.872 36.654 1.0026.02 C ATOM 5175 CAC HEM 605 32.277 10.303 38.134 1.00 28.75 C ATOM5176 CBC HEM 605 32.723 11.023 39.147 1.00 29.46 C ATOM 5177 ND HEM 60530.822 7.568 33.762 1.00 24.98 N ATOM 5178 C1D HEM 605 30.449 7.69035.064 1.00 26.97 C ATOM 5179 C2D HEM 605 29.334 6.838 35.372 1.00 27.95C ATOM 5180 C3D HEM 605 29.022 6.186 34.199 1.00 28.91 C ATOM 5181 C4DHEM 605 29.960 6.655 33.195 1.00 26.41 C ATOM 5182 CMD HEM 605 28.6586.696 36.726 1.00 29.28 C ATOM 5183 CAD HEM 605 27.918 5.166 34.005 1.0030.52 C ATOM 5184 CBD HEM 605 28.517 3.738 34.229 1.00 33.73 C ATOM 5185CGD HEM 605 27.558 2.594 34.062 1.00 36.10 C ATOM 5186 O1D HEM 60527.962 1.415 34.024 1.00 36.59 O ATOM 5187 O2D HEM 605 26.323 2.85733.954 1.00 38.61 O ATOM 4670 OH2 WAT 1040 35.718 9.520 42.672 1.0042.18 O ATOM 4688 OH2 WAT 1058 28.964 10.656 33.920 1.00 22.72 O ATOM4689 OH2 WAT 1059 30.899 0.748 34.525 1.00 20.23 O ATOM 4729 OH2 WAT1099 27.275 9.551 36.040 1.00 39.65 O ATOM 4758 OH2 WAT 1128 26.3733.276 37.398 1.00 47.19 O ATOM 4782 OH2 WAT 1152 25.763 5.450 39.6951.00 46.21 O ATOM 4874 OH2 WAT 1244 25.312 6.991 36.325 1.00 42.15 OATOM 4888 OH2 WAT 1258 22.019 −1.420 −22.953 1.00 31.72 O ATOM 5039 OH2WAT 1409 31.245 2.148 31.930 1.00 37.70 O ATOM 5051 OH2 WAT 1421 27.033−0.096 31.578 1.00 53.89 O END

1. An isolated heme/albumin complex having an atomic arrangement of coordinates comprising the coordinates as set forth in Appendix A.
 2. A complex of hemalbumin having a crystal form of the space group C2 and unit cell dimensions of a=183.11 Å, b=37.91 Å, c=94.83 Å, and β=105.04°.
 3. A complex of hemalbumin according to claim 2 wherein the active site comprises a hydrophobic surface of the binding pocket comprising amino acid residues Tyr-161, Phe-157, Arg-186, Leu-182, Arg-117, Phe-134, Leu-135, Leu-154, Phe-149, Ile-142, His-146, Arg-114, Lys-190, Ser-193, Ala-158, Tyr-138, Leu-115, Met-123, Phe-165, and Pro-118.
 4. A method for obtaining a high-resolution set of atomic coordinates of hemalbumin comprising preparing a crystal of hemalbumin suitable for X-ray diffraction, obtaining an X-ray diffraction pattern of said crystal using X-rays or neutrons, and resolving said X-ray diffraction pattern to a resolution of at least 1.9 Angstroms so as to obtain a high resolution set of atomic coordinates of hemalbumin.
 5. The method of claim 4 wherein said X-ray diffraction pattern is resolved using an array of CCD detectors to obtain a plurality of oscillation images of the crystal.
 6. A set of atomic coordinates of a hemalbumin crystal obtained by the method of claim
 4. 7. A method of increasing the gas-binding properties of heme or albumin comprising obtaining a high-resolution structure of the atomic coordinates of the heme/albumin binding complex, examining said structure to determine the location of binding residues of heme or albumin for a particular gas, and substituting at least one binding residue of heme or albumin with a residue having increased binding affinity for a particular gas.
 8. The method according to claim 7 wherein the gas is selected from the group consisting of oxygen, cyanide, nitric acid and carbon monoxide.
 9. A method of obtaining a blood substitute which can reversibly bind oxygen comprising obtaining a high-resolution structure of the atomic coordinates of the heme/albumin binding complex, examining said structure to determine the binding residues of heme or albumin involved in the binding of oxygen, and substituting at least one binding residue of heme or albumin with a residue having increased binding affinity for oxygen.
 10. A blood substitute which can reversibly bind oxygen obtained by the method of claim
 9. 11. A recombinant serum albumin having a mutation in an amino acid residue selected from the group consisting of Tyr-161, Phe-157, Arg-186, Leu-182, Arg-117, Phe-134, Leu-135, Leu-154, Phe-149, Ile-142, His-146, Arg-114, Lys-190, Ser-193, Ala-158, Tyr-138, Leu-115, Met-123, Phe-165, and Pro-118, wherein such mutation increases the gas binding affinity of said albumin.
 12. The recombinant serum albumin according to claim 11 wherein the mutation is the substitution of a hydrophilic residue.
 13. The recombinant serum albumin according to claim 11 wherein the gas binding affinity increased by the mutation is oxygen binding affinity.
 14. An isolated nucleic acid fragment coding for the albumin of claim
 11. 15. A method of obtaining heme derivatives with increased oxidative stability and an increased capacity to reversibly bind oxygen comprising obtaining a high-resolution structure of the atomic coordinates of the heme/albumin binding complex, examining said structure to determine the binding residues of heme involved in the binding of oxygen, and substituting at least one binding residue of heme with a residue having increased oxidative stability sufficient to increase its capacity for reversibly binding oxygen.
 16. A heme derivative obtained by the method of claim
 15. 17. An isolated albumin fragment comprising the heme binding domain IB.
 18. An isolated polymer or fusion protein containing the heme binding domain IB.
 19. A method of imparting heme binding properties in a non-human albumin comprising substituting lysine for leucine at the amino acid position equivalent to position 190 of human serum albumin as determined by sequence alignment.
 20. The method according to claim 19 wherein the albumin is non-human mammalian albumin. 